Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with Supernatant pig heart enzyme.

Supernatant malate dehydrogenase from pig heart, a dimeric protein containing two very similar or identical subunits, shows negatively cooperative (anticooperative) interactions between NADH binding sites in the presence, but not in the absence, of 0.1 M L-malate. This behavior is observed consitently whether the technique used employs protein fluorescence quenching, NADH fluorescence enhancement, or ultrafiltration dialysis. Fluorescence titration shows that L-malate is also anticooperatively bound in the presence of saturating concentrations of NADH. The data are consistent with an "induced asymmetry" model in which conformational change accompanies the formation of the ternary complex. Two of the three chromatographically resolvable forms of the enzyme have been tested and found to have anticooperative behavior.


The role of arginyl residues in directing carboxymethylation of horse liver alcohol dehydrogenase.

The selective carboxymethylation by iodoacetate of Cys-46 in the active center of horse liver alcohol dehydrogenase has been shown to be mediated by interaction of the anionic reagent with the arginyl residue(s) previously shown to be responsible for binding NADH (L.G. Lange, J.F. Riordan, and B.L. Vallee (1974), Biochemistry 13, 4361). Thus, sequential and reversible chemical modification of arginine with butanedione and of cysteine with pmercuribenzoate demonstrate that the essential thiol groups are not affected by arginine modification. Importantly, the rate of incorporation of [14C]idoacetate into native horse liver alcohol dehydrogenase is ten times faster than that for the butanedione-modified enzyme. Moreover, as evidenced by peptide isolation, the radiolabel incorporated into the latter occurs at low levels in several different peptides as opposed to the single, strongly labeled CmCys-46 peptide obtained from the native enzyme. The demonstration that the arginyl residue(s) involved in coenzyme binding promotes enhanced reactivity of the active site thiol supports the general hypothesis that the spatial arrangement of structural features allowing expression of enzymatic function may also account for enhanced chemical reactivity of certain active site residues (B.L Vallee and J.F. Riordan (1969), Annu. Rev. Biochem. 38, 733).


Nucleotide regulation of a eukaryotic protein synthesis initiation complex;.

Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.


Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+.  Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.


EPR studies on compound I of horseradish peroxidase.

Compound I of horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase EC 1.11.1.7) was studied by EPR at low temperatures. An asymmetric signal was found, about 15 Gauss wide and with a g-value of 1.995, which could be detected only at temperatures below 20 K and which had an intensity corresponding to about 1% of the heme content. In a titration with H2O2, the signal intensity was proportional to the concentration of Compound I, reaching a maximum when equivalent amounts of H2O2 were added. This indicates that the signal is not due to an impurity, and it is suggested that a free radical is formed, relaxed by a near-by fast-relaxing iron.


Isolation of an activator of multiple forms of cyclic nucleotides phosphodiesterase of rat cerebrum by isoelectric focusing.

An isoelectric focusing technique was used to isolate multiple forms of cyclic nucleotide phosphodiesterase from a 105 000 times g soluble supernatant fraction of sonicated rat cerebrum. These separated peaks of activity had iso-electric points of 5.1, 5.6, 6.0, 6.6, 8.0, and 9.0. The activities were not stimulated by an endogenous activator of the enzyme but were inhibited by EGTA treatment. However, activator-sensitive forms of the enzyme could be separated from brain if the preparation of rat cerebrum was dialyzed against an EGTA containing buffer prior to electrofocusing. The procedure was also used to isolate a column fraction that stimulated maximum velocities of cyclic AMP and cyclic GMP hydrolysis. This fraction was itself devoid of phosphodiesterase activity and had an isoelectric point of 4.7.


Characterization of human alpha-galactosidase A and B before and after neuraminidase treatment.

It has been previously reported that following neuraminidase treatment alpha-galactosidase A is converted into the B form, as revealed by electrophoresis. By a variety of techniques such as isoelectrofocusing, DEAE-chromatography and by enzyme kinetic parameters, no conversion of alpha-galactosidase A into B, or the reverse, could be detected after neuraminidase treatment. Only an apparent transformation of alpha-galactosidase A into B was revealed by Cellogel electrophoresis. In addition, a discrepancy was noticed between the pattern of electrophoretic migration on starch gel and Cellogel and the net electrical charges of the two alpha-galactosidases as deduced by isoelectrofocusing and DEAE-cellulose. Neuraminidase treatment did not affect the activity of alpha-galactosidase A towards the natural substrate, ceramidetrihexoside, but the activity of alpha-galactosidase B decreased by about 30% under the same conditions. The two forms of alpha-galactosidases A and B used in this study were extensively purified by classical procedures.


Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.


Primary structure of cytochrome c from the insect Ceratitis capitata.

The complete amino acid sequence of cytochrome c from the Dipterous Ceratitis capitata (serie Acalypterae) has been determined by combining automatic and manual methods of sequence analysis. No overlaps between positions 79 and 80, 86 and 87, 91 and 92 as well as between 99 and 100 were obtained. The alignment of these peptides was done by homology with other sequences of cytochromes c from insects already described. Comparison with the sequences of cytochromes c of other Diptera studied so far shows three changes (positions 50, 60 and 61, according to vertebrate cytochrome c numeration) from the Acalypteran Drosophila melanogaster and five changes (positions 9, 36, 50, 60 and 61) from that of the Calypteran Haematobia irritans.


The demonstration of ferrihemochrome intermediates in heinz body formation following the reduction of oxyhemoglobin A by acetylphenylhydrazone.

Interaction of acetylphenylhydrazine with oxyhemoglobin A in a hemolysate or in intact red cells resulted in the formation of ferrihemochromes as shown by a characteristic optical spectrum. The same optical spectrum was observed in a suspension of red cell ghosts containing numerous Heinz bodies. Electron paramagnetic resonance of actylphenylhydrazine-incubated red cells disclosed the presence of previously identified reversible ferrihemochromes, which can be reduced to functional hemoglobin, and irreversible ferrihemochromes, which cannot be reduced to functional hemoglobin. (Ferrihemochromes are defined as low spin forms of ferric hemoglobin having heme ligands endogenous to the protein structure). In contrast, only irreversible ferrihemochromes could be observed in ghosts containing Heinz bodies. In addition both optical and magnetic features of sulfhemoglobin were observed in an acetylphenylhydrazine-treated red cell hemolysate. Similar optical features are produced by the interaction of aromatic nitrogen-containg reductants with purified oxyhemoglobin in the presence of (NH4)2S. This reaction is not effected by the presence of catalase, suggesting that H2O2 is not an intermediate of the reaction. It is concluded that the mechanism of action of acetylphenylhydrazine with oxyhemoglobin is two-fold, ultimate reduction to high spin ferric hemoglobin followed by ferrihemochrome formation. Thus it appears that the pathway of denaturation of hemolytic anemias and thalassemia or induced by chemical reagents, entails a common route involving the formation of ferric hemoglobin by a reductive mechanism, followed by reversible ferrihemochromes, irreversible ferrihemochromes, and ultimately, precipitation.


Isolation and partial characterization of the major apolipoprotein component of bovine serum high density lipoprotein.

The delipidated protein component of bovine serum high density lipoprotein was fractionated by gel filtration on a Sephadex G-150 column (equilibrated with buffer containing 6 M urea) into three fractions: I, II and III. Fractions I and II together constitute 88% of all the protein weight of bovine high density lipoprotein, whereas fraction III accounts for the remaining 12%. Analysis of the fractions by sodium dodecyl sulfate-polyacrylamide electrophoresis reveals that fraction I consists mostly of aggregated forms of fraction II and some higher molecular weight species, probably irreversible aggregates of fraction II. The irreversible aggregates are apparently formed during the delipidation procedure or upon aging of the lipoprotein. The major protein component of the high density bovine lipoprotein is found in fraction II; it has a molecular weight of 27 000 plus or minus 1500 and appears to be homogeneous by several physicochemical criteria. The amino acid composition of fractions I and II are essentially identical; their spectral properties, including absorption, fluorescence, and circular dichroism spectra, are similar; however, fraction I appears to contain traces of oxidized lipid and more secondary alpha-helical organization than fraction II. By comparison with the intact lipoprotein, which contains about 65% of alpha-helical structure, fractions I and II have diminished alpha-helical organization, 55% and 43%, respectively. Fraction III, on sodium dodecyl sulfate-polyacrylamide electrophoresis, separates into two protein bands of equivalent intensity, having molecular weights around 13 000 and 11 000. Fraction III is markedly distinct from the other two, in amino acid composition and spectral properties, especially in its red-shifted fluorescence and very low content of alpha-helical structure. The protein composition of bovine serum high density lipoprotein is compared with recently published results for high density lipoprotein apoproteins of man, chimpanzee, rhesus monkey, pig and rat. Similarities and differences are discussed in terms of possible evolutionary and functional factors.


Self-association of the major protein component of bovine serum high density lipoprotein.

The major protein component of bovine high density lipoprotein was investigated in solution by fluorescence polarization and ultracentrifugal techniques. A fluorescent derivative of this protein with 1-dimethylaminonaphthalene-5-sulfonyl chloride was employed in the fluorescence experiments. Over the concentration range from 5-10(-7) M to 5-10(-4) M of the protein monomer at pH values from 2 to 11 and ionic strengths from 0.03 to 2.0, at 23 degrees C, the major protein of bovine high density lipoproteinapoprotein (Apo-HOL-I) was found to exist in a stable aggregated form. The aggregate was not affected by dioxane additions of up to 20% nor by Triton X-100 to 0.2%, but dissociated readily in the presence of 0.07% sodium dodecylsulfate or 6 M urea. At concentrations below 5-10(-7) M, dissociation of the protein aggregate started spontaneously and continued down to 10(-8) M, the lowest measurable concentration. Several physiocochemical properties of the major protein of bovine high density lipoprotein were determined in the stable aggregate form. Molecular weight was 104 000 from ultracentrifugal analysis and 80 000 from gel-filtration. Rotational relaxation time was 115 ns at 25 degrees C, and s-0 20,w was 4.78 s. The results suggest very strong protein-protein interactions (Kd less than 10(-7) M) that are not electrostatic in nature. Hydrophobic interactions of a magnitude that could be affected by 20% dioxane or 0.2% Triton X-100 detergent are also excluded. There is saturation of the interaction sites by the aggregation of a few protein monomer units possibly to form a tetramer which is moderately asymmetric (1:4 axial ratio, assuming an ellipsoid of revolution) and relatively rigid. The strong protein-protein interactions in this pure apolipoprotein suggest the possibility of competition of inter-protein associations with protein-lipid interactions in in vitro lipid binding or lipoprotein reconstitution experiments.


Effects of local anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian cells to agglutination by plant lectins.

Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface.


Bile salt alteration of ion transport across jejunal mucosa.

The effect of conjugated dihydroxy and trihydroxy bile salts on electrolyte transport across isolated rabbit jejunal mucosa was studied. Both taurochenodeoxycholic acid and taurocholic acid increased the short-circuit current (Isc) in bicarbonate-Ringer solution but not in a bicarbonate-free, chloride-free solution. Taurochenodeoxycholic acid was significantly more effective than taurocholic acid in increasing Isc. The presence of theophylline prevented the taurochenodeoxycholic acid- and taurocholic acid-induced increase in Isc. Transmural ion fluxes across jejunal mucosa demonstrated that 2 mM taurochenodeoxycholic acid decreased net Na+ absorption, increased net Cl- secretion and increased the residual flux (which probably represents HCO3- secretion). These studies support the hypothesis that cyclic AMP may be a mediator of intestinal electrolyte secretion.


Interaction of lysine-rich histone and DNA in chromatin structure.

The heat denaturation and renaturation curves of rat liver and ascites hepatoma (AH 108A) chromatins were measured. In these renaturation curves, there are small sigmoidal regions. These sigmoidal regions remained in redenaturation curves and were largely stable to DNAase I digestion. When the chromatins were treated stepwise with NaClO4 and lysine-rich histones were removed, the sigmoidal regions in the renaturation curves disappeared. These results suggested that the sigmoidal regions reflected the interaction of DNA and lysine-rich histones.


Non-histone chromosomal proteins from virus-transformed and untransformed 3T3 mouse fibroblasts.

A peak in the non-histone chromosomal protein polyacrylamide gel electrophoresis profiles has been detected which is higher in log phase 3T3 and 3T3/SV40 cells than in density-inhibited 3T3 cells. Radioactive incorporation is substantially higher into this peak in log phase 3T3 than in 3T3/SV40 and density-inhibited 3T3 cells. Reversion of 3T3/SV40 cells with dibutyryl cyclic AMP and theophylline produces increased radioactive incorporation into the peak. Electrophoresis of non-histone chromosomal proteins extracted at different stages of the cell cycle in density inhibited 3T3 cells following serum stimulation shows a cyclic variation in the amount of this peak with maximum accumulation in late G1. In contrast the height of an equivalent peak in synchronously growing 3T3/SV40 cells remains constant throughout the cell cycle. It is postulated that the protein(s) of this peak may have a regulatory role in cell growth.


DNA synthesis in L-cells in the presence of 5-fluorodeoxyuridine.

After 16 h of incubation with 10-minus6 M FdUrd, the rate of (32P) orthophosphate uptake into DNA isolated from L-cells amounted to 15% of that of an untreated culture, although cell division had stopped several hours earlier. All 4 deoxynucleotides were present in this DNA but its nucleotide composition, as measured by enzymatic digestion and chromatography, reflected a decreased thymidine precursor pool in FdUrd-treated cells. Sedimentation analysis in alkaline sucrose gradients revealed that the DNA formed in the presence of FdUrd had a sedimentation coefficient of 10 S which corresponded to a single-stranded molecular weight of 5.5.105. This DNA could be "chased" into a high molecular weight DNA if the FdUrd block was bypassed with added dThd or BrdUrd. Other analyses failed to detect RNA covalently linked to the DNA fragments at a level of more than 5% RNA or about 90 ribonucleotides. The accumulation of these DNA fragments could be explained by assuming that in the presence of limiting precursor pool the rate of DNA chain initiation is greater than the rate of chain elongation.


A cell-free mammalian protein-synthesizing system stimulated by RNA from avian myeloblastosis virus.

Carcinoma cells, oncornavirus-infected cells and fetal bovine tissue provide salt wash ribosomal factors capable of responding to avian myeloblastosis virus (AM virus)-RNA and stimulating the incorporation of amino acids into proteins as well as catalyzing the binding of N-acetylated (35S) methionyl-tRNA. The exogenously dependent amino acid incorporation system is stimulated by the high molecular weight species of AM virus-RNA only, particularly the fraction containing polyadenylate (poly(A)) residues; the system is also markedly inhibited by the low molecular weight AM virus-RNA species. Activity for the exogenous system displays very definite divalent/monovalent cation optima and requires the presence of mammalian transfer RNA.


The oxidation of tiron by superoxide anion. Kinetics of the reaction in aqueous solution in chloroplasts.

The rate of reaction between superoxide anion (O2) and 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron) was measured with pulse radiolysis-generated O2. A kinetic spectrophotometric method utilizing competition between p-benzoquinone and tiron for O2 was employed. In this system, the known rate of reduction of p-benzoquinone was compared with the rate of oxidation of tiron to the semiquinone. From the concentration dependence of the rate of tiron oxidation, the absolute second order rate constant for the reaction was determined to be 5x10-8 M-minus1-s-minus1. Ascorbate reduced O2 to hydrogen peroxide with a rate constant of 10-8 M-minus1-s-minus1 as determined by the same method. The tiron semiquinone may be used as an indicator free radical for the formation of superoxide anion in biological systems because of the rapid rate of oxidation of the catechol by O2 compared to the rate of O2 formation is most enzymatic systems. Tiron oxidation was used to follow the formation of superoxide anion in swollen chloroplasts. The chloroplasts photochemically reduced molecular oxygen which was further reduced to hydrogen peroxide by tiron. Tiron oxidation specifically required O2 since O2 was consumed in the reaction and tiron did not reduce the P700 cation radical or other components of Photosystem I under anaerobic conditions.


Electron transport in the membrane of lutoids from the latex of Hevea brasiliensis.

1. An antimycin-insensitive NADH-cytochrome c oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex of Hevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH. 2. Two beta-type cytochromes are present in the membranes. Cytochrome beta563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 degrees K. A second cytochrome, cytochrome beta561, seems to be reducible by hydrosulfite only. 3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochrome P-450 could not be demonstrated. 4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.


Energy-linked and energy-independent transhydrogenase activities in Escherichia coli vesicles.

Active transport vesicles of Escherichia coli were shown to possess low levels of energy-independent and energy-dependent nicotinamide nucleotide transhydrogenase activities. Breakage of such vesicles in a French pressure cell resulted in a fraction which had an 8-10-fold increased respiration- and ATP-driven transhydrogenase activities. Stimulation of the ATPase activity in vesicles with Triton X-100 was also paralledled by a 2-fold increase in the energy-independent transhydrogenase. Disruption of the vesicles similarly resulted in increases in the energy-independent transhydrogenase, NADH and succinate oxidase activities but a decrease in succinate supported proline uptake. In the light of these findings, the "sidedness' of the vesicle membranes is discussed.


Two electron donation sites for exogenous reductants in chloroplast photosystem II.

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20-40%) in Signal IIf magnitude accompanying donor addition at low (smaller than 10(-%) M) concentrations, but the extent of the decrease does not change further with increasing donor concentrations. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration. In the presence of the hydrophilic donor, Mn-2+, the Signal IIf decay halftime remains constant with increasing Mn-2+ concentration. However, the flash-induced Signal IIf magnitude pregressively decreases with increasing Mn-2+ concentration. These results are interpreted in terms of two competing paths for the reduction of P680+. In one path P680+ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn-2+ reduces P680+ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.


Nature of photochemical reactions in chromatophores of Chromatium D. III. Heterogeneity of the photosynthetic units.

The effect of isooctane extraction on photooxidation of c-type cytochromes was investigated in Chromatium chromatophores. Photooxidation of cytochrome c-555 was not affected by isooctane-extraction was abolished by thorough extraction of ubiquinone-7, but the quantum yield of the cytochrome photooxidation remained unchanged until 90% of the total ubiquinone was extracted. The photooxidation of cytochrome c-552 was recovered by the addition of ubiquinone-7 but not by menaquinone. A dark incubation of sufficient length was needed for maximal quantum yield of cytochrome c-555 photooxidation in the presence of 30 mM ascorbate. It is proposed that there are two types of photosynthetic units (or associations of molecules involved in the primary redox reactions) in Chromatium chromatophores. The combinations of primary electron donor-reaction center chlorophyll-primary electron acceptor may be cytochrome c-552-P890=ubiquinone in one type and cytochrome c-555-P890-X in another.


Stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase from Pseudomonas fluorescens.

The stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase (E.C. 1.1.1.48) from P. fluorescens was investigated. The label at C-1 of D-[1--3H] galactose was enzymatically transferred to NAD and the resulting [4--3H]NADH was isolated and its stereochemistry at C-4 investigated. It was found that the label was exclusively located at the 4(S) position in NADH which calls for classification as a B-enzyme. This result was confirmed by an alternate approach in which [4--3H]NAD was reduced by D-galactose as catalyzed by D-galactose dehydrogenase. The sterochemistry at C-4 of the nicotinamide ring would then have to opposite to that in the first experiment. As expected, the label was now exclusively located in the 4(R) position, again confirming the B-calssification of the enzyme.


Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.


Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis.

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [ (S)alanine: NAD oxidoreductase (EC 1.4.1.1) ] from B. subtilis was investigated. The label at C-2 of (S) [2,3--3H] alanine was enzymatically transferred to NAD, and the [4--3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4--3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the ]4--3H]NADH produced. As expected, the label was now exclusively located at the (S) position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.


The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue.

1. The acyl-CoA synthetase activity in brown adipose tissue of cold-exposed guinea pig has been studied by measuring the rate of palmitoylcarnitine formation in the presence of excess carnitine palmitoyltransferase. 2. The rate of palmitoylcarnitine formation in the mitochondria was found to be 161 plus or minus 64 nmol.mg-minus-1. min-minus-1 (n=9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 plus or minus 1 nmol endogenous fatty acids.mg-minus-1 were activated with three different mitochondrial preparations. 4. Three different experimental approaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (B) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark. T. (1973) Anal. Biochem. 56, 324-340) and (c) the determination of the stoichiometry between the activities of these enzymes sedimented at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.


Effects of several common long chain fatty acids on the properties and lipid composition of the very low density lipoprotein secreted by the perfused rat liver.

1. Livers from normal fed male rats were perfused in vitro with a bloodless medium which contained intially 3% bovine serum albumin and 100 mg% glucose. Albumin alone, or myristate (14 : 0), palmitate (16 : 0), palmitoleate (16 : 1), stearate (18 : 0), oleate (18 : 1), or linoleate (18:2) was infused at a constant rate (496 mumol/4 h), as a complex with albumin, during the experiment. 2. The very low density lipoprotein secreted by the liver after infusion of unsaturated fatty acids (16 : 1, 18 :1, 18 : 2) has a faster rate-zonal mobility in the ultracentrifuge and is, therefore, probably a larger particle with fewer moles of phospholipid and cholesterol relative to triacyglycerol (triacyglycerol/phospholipids/cholesterol = 100/25.1/16.4) than the very low density lipoproteins produced after infusion of saturated (14 : 0, 16 : 0, 18 : 0) fatty acids (triacyglycerol/phospholipids/cholesterol = 100/30.1/19.1). The molar ratio of phosphoipids/cholesterol of the very low density lipoprotein was similar regardless of which fatty acid was infused. The predominant fatty acid of the very low density lipoprotein or hepatic triacyglycerol, in all cases, was the infused acid. 3. We conclude that free fatty acid regulates the quantity and proportions of triacyglycerol, phospholipids, and cholesterol secreted by the liver in the very low density lipoprotein, and therefore, may secondarily influence concentrations of lipids in the very low density lipoprotein and other plasma lipoproteins circulating in vivo.


The effects of hyperlipidemia on lipoprotein metabolism in squirrel monkeys and rabbits.

We studied the metabolism of different classes of lipoprotein in squirrel monkeys and rabbits. Lipoproteins were labeled in vivo in donor animals with (3H)leucine and (3H)cholesterol. The rate of disappearance from plasma of recipient squirrel monkeys of the protein moiety of the very low density lipoproteins was rapid, that of high density lipoproteins slow, and the rate for low density lipoproteins was intermediate. The fractional turnover of the apoprotein of low density lipoproteins was slightly reduced in hyperlipidemic monkeys, but the absolute rates of synthesis and catabolism were increased. Hyperdipidemia in rabbits resulted in a dramatic reduction in the fractional catabolic rate of low density lipoprotein apoprotein. Hyperlipidemia in the donors of biosynthetic low density lipoproteins also influenced the rates of catabolism in rabbits. We showed the cycloheximide that although there was recycling of (3H)leucine into other proteins, the reutilization of leucine from low density lipoproteins for nascent low density lipoproteins was not significant. In most tissues the ratio of cholesterol:protein radioactivity was much greater than that for plasma 24 h after administration of labeled low density lipoproteins, but the ratios for aortic intima plus inner media and for plasma low density lipoproteins were similar. The presence of atherosclerosis resulted in a large increase in the apparent uptake of low density lipoproteins by the aortas of rabbits and monkeys.


Induction of cystathionase in foetal rat liver explants. Effects of dexamethasone, N-6, O-2 -dibutyryladenosine 3,5 -monophosphate and glucagon in vitro.

At the 18th day of gestation and thereafter foetal rat liver explants in organ culture showed the competence to respond to dexamethasone by increased cystathionase activity, whereas the ability to respond to dibutyryl cyclic AMP or glucagon became evident at a later developmental stage (during the last 2 days prior to term). Simultaneous incubation with cycloheximide inhibited the stimulatory effect of these agents on foetal rat liver cystathionase activity in vitro. Dexamethasone and glucagon were both capable of increasing liver cystathionase activity both in newborn and 3-day-old animals in vivo.


Reduced glucocorticoid responsiveness and receptor concentration in splenic leukocytes of senescent rats.

Glucocorticoids are 63% less effective in inhibiting [5-3-h] uridine uptake by splenic leukocytes of senescent rats (24-14 months) than by those of mature adult animals (12-14 months). Scatchard analyses reveal 57% fewer specific glucocorticoid binding sites in cytosols of the senescent group. Reduced binding of cortisol by cytosol macromolecules of intact viable leukocytes at 37 degrees C is also apparent in the older rats. Specific binding of glucocorticoids to cytoplasmic macromolecules appears to be closely linked, if not required, for inhibition of [5-3-hi1 uridine uptake. This is evidenced by the fact that both inhibition and specific binding are simultaneously blocked by progesterone administration. In addition, the amount of specific binding as well as the degree of inhibition of [5-3-h] uridine uptake is concentration dependent up to 2. 10-6 M cortisol. Thus, decreased glucocorticoid reponsiveness in the cells from senescent rats appears to be due primarily to decreased concentrations of specific glucocorticoid receptors.


Modifying actions of calcium ionophores on insulin release.

Beta-Cell-rich pancreatic islets were microdissected from noninbred ob/obmice and exposed to the calcium ionophores X-537A and A-23187. X-537A differed from A-23187 in being a potent insulin secretagogue at non-stimulating glucose concentrations. Both ionophores inhibited the stimulation of insulin release obtained after adding 20 mM glucose to the incubation medium. The latter observation is consistent with the idea of a reduced beta-cell function when the Ca-2+ in the functionally important intracellular pool (s) exceeds a certain concentration. The ionophore inhibition of the glucose-stimulated insulin release may at least in part result from decreased formation of cyclic AMP, since X-537A proved to be as effective as L-epinephrine in reducing the islet content of this nucleotide in the presence of a phosphodiesterase inhibitor. The secretagogic action of X-537A at a low glucose concentration persisted when different ions were omitted from the incubation medium and was actually considerably enhanced in the absence of extracellular Ca-2+. The insulin-releasing action of X-537A was neither influenced by 3-O-methyglucose nor by drugs blocking the alpha or beta-adrenergic receptor sites. Exposure of the pancreatic beta-cells to metabolic inhibitors in concentrations which significantly reduced the secretory response to glucose, potentiated stimulation of insulin release by X-537A, suggesting that this effect may in part be accounted for by intracellular dissolution of secretory granules.


Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol L-serrine. Pyruvate aminotransferase by dibutyryl adenosine 3,5-monophosphate.

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutyryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine:pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have not effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serin aminotransferase diminishes with the age of animall. Increases in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyryl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.


On the location of the sulfhydryl group in bovine plasma albumin.

A series of spin labels has been empolyed to explore the environment of the sulfhydryl group in bovine plasma albumin. The spin labels consist of the nitroxide-free radical and a maleimide (or iodoacetamide)-attaching group separated by varying chain lenghths. Both sets of spin labels preferentially bind to the sulfhydryl group under appropriate conditions. From the change in the electron spin resonance spectra of these nitroxides as a function of chain length, we conclude that the sulfhydryl group is located in a crevice approx. 9.5 A in depth.


The application of collagenase, purified by affintiy chromatography, to the isolation of insoluble elastin from bovine aorta.

Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.16), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine aorta. The extremely low level of N-terminal residues (1.6 mol per 10(6) g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. The amino acid profile of the aortic elastin was found to be almost identical to that of insoluble elastin prepared from bovine ligamentum nuchae by the same method.


Nitrogenase. VIII. Mössbauer and EPR spectroscopy. The MoFe protein component from Azotobacter vinelandii OP.

We have studied the molybdenum-iron protein (MoFe protein, also known as component I) from Azobacter vinelandi using Mössbauer spectroscopy and electron paramagnetic resonance on samples enriched with 57Fe. These spectra can be interpreted in terms of two EPR active centers, each of which is reducible by one electron. A total of four different chemical environments of Fe can be discerned. One of them is a cluster of Fe atoms with a net electronic spin of 3/2, one of them is high-spin ferrous iron and the remaining two are iron in a reduced state (probably in clusters). The results are as follows: Chemical analysis yields 11.5 Fe atoms and 12.5 labile sulfur atoms per molybdenum atom; the molecule contains two Mo atoms per 300 000 daltons. The EPR spectrum of the MoFe protein exhibits g values at 4.32, 3.65 and 2.01, associated with the ground state doublet of a S = 3/2 spin system. The spin Hamiltonian H = D(S2/z minus 5/4 + lambda(S2/x minus S2/y)) + gbeta/o S-H fits the experimental data for go = 2.00 and lambda = 0.055. Quantitative analysis of the temperature dependence of the EPR spectrum yields D/k = 7.5 degrees K and 0.91 spins/molybdenum atom, which suggests that the MoFe protein has two EPR active centers. Quantitative evaluation of Mössbauer spectra shows that approximately 8 iron atoms give rise to one quadrupole doublet; at lower temperatures magnetic spectra, associated with the groud electronic doublet, are observed; at least two magnetically inequivalent sites can be distinguished. Taken together the data suggest that each EPR center contains 4 iron atoms. The EPR and Mössbauer data can only be reconciled if these iron atoms reside in a spin-coupled (S = 3/2) cluster. Under nitrogen fixing conditions the magnetic Mössbauer spectra disappeared concurrently with the EPR signal and quadrupole doublets are obserced at all temperatures. The data suggest that each EPR active center is reduced by one electron. The Mössbauer investigation reveals three other spectral components characteristic of iron nuclei in an environment of integer or zero electronic spin, i.e. they reside in complexes which are "EPR-silent". One of the components (3-4 iron atoms) has Mössbauer parameters characteristic of the high-spin ferrous iron as in reduced ruberdoxin. However, measurements in strong fields indicate a diamagnetic environment. Another component, representing 9-11 iron atoms, seems to be diamagnetic also. It is suggested that these atoms are incorporated in spin-coupled clusters.


A critical evaluation of the proposal that serum apolipoproteins are the major constituents of the human erythrocyte membrane.

1. The EDTA and Triton X-100 extracts of human erythrocyte ghosts gave no precipitin lines in double diffusion analyses with antibodies to either lipoprotein A, lipoprotein B, lipoprotein C, lipoprotein D, Lp(a) lipoprotein or arginine-rich apolipoprotein of normal human serum (for nomenclature for serum lipoprotein families and apolipoptoteins, see Alaupovic, P., Kostner, G., Lee, D. M., McConathy, W.J. and Magnani, HN. (1972) Expo. Annu. Biochem. Med. 31, 145-160 and Alaupovic, P., Lee, D.M. and McConathy, W.J., (1972) Biochim. Biophys. Acta 260, 689-707.) These membrane preparations also reacted negatively with commercially available antisera to alpha- and beta-lipoproteins. 2. The normal serum very low density, low density and high density lipoproteins formed no precipitin lines with antibodies to either intact or EDTA-extracted ghosts. 3. The serum apolipoproteins and their constitutive polypeptides (A-I, A-II, B, C-I, C-II, C-III, D and arginine-rich apolipoprotein) reacted negatively with antibodies to intact or EDTA-extracted ghosts. The EDTA and Triton X-100 extracts of erythrocyte ghosts gave no reaction with monospecific antibodies to serum apolipoproteins and their constitutive polypeptides. 4. Ghosts dissolved 2% sodium dodecyl sulfate gave positive immunoprecipitin lines with antisera to alpha- and beta-lipoproteins. However, the sodium dodecyl sulfate solution in concentrations greater than 0.1% also formed precipitin lines with antisera to the same lipoproteins. 5. These results do not support the suggestion (Langdon, R.G. (1974) Biochim. Biophys. Acta 342, 213-228) that serum apolipoptoteins are integral protein constituents of human erythrocyte ghosts. The immunoprecipitin lines observed in the latter study might have been due to the presence of trace amounts of serum lipoproteins loosely attached to the cellular surfaces or, more probably, resulted from nonspecific interactions between the proteins and the sodium dodecyl sulfate used as the solubilizing agent


[Photochemical generation of the cation-radical of bacteriochlorophyll b and the anion-radical of bacteriochlorophyll b and bacteriopheophytin b, components of the photosynthesizing bacteria Rhodopseudomonas viridis].

The mechanism of photochemical redox reaction of bacteriochlorophyll b, bacteriopheophitin b was studied. The oxidation of bacteriochlorophyll b was carried out in ethanole by means of p-benzoquinone. In ESR spectra the singlet line with delta H = 8.4 gs and g = 2.0025 corresponded to cation-radical. The changes within the region of 400-500 nm, 500-600 nm and the band with the maximum at 685 nm corresponded to cation-radical in the absorption spectra. Photoreduction of bacteriochlorophyll b and bacteriopheophitin b was carried out in triet containing phenilhydrosin. Anion-radicals of bacteriochlorophyll b and bacteriopheophitin b observed in the ESR spectra had singlet form with weak and superthin structure and correspondently with delta H = 10 gs, delta H = 12 gs. The spectrophotometric characteristics of the above pigments were obtained.


Presence of the methylester of 5-carboxymethyl uridine in the wobble position of the anticodon of tRNAIII Arg from brewer's yeast.

The methylester of 5-carboxymethyluridine (mcm5U), its degradation product 5-carboxymethyluridine (cm5U) and the corresponding nucleotide (cm5Up) were isolated from brewer's yeast tRNAIII Arg or from the dodecanucleotide containing the anticodon. Their chromatographic and electrophoretic properties and their UV absorbing spectra were identical to that of the corresponding synthetic compounds. The gas chromatographic behavior and the mass spectrum of mcm5U obtained from tRNAIII Arg and of a synthetic sample were also identical ; the rare occurence of a thermal reciprocal bimolecular methyl-hydrogen transfer in the mass spectrometer ion source was observed. A mild alkaline treatment of tRNAIII Arg leads to the saponification of mcm5U into cm5U (within the tRNA), which can be again esterified in the presence of a yeast homogenate and (methyl-14C) S adenosylmethionine. The radioactivity was found in the mcm5U located in the wobble position of the anticodon of tRNAIII Arg. The presence of this odd nucleotide in that position could possibly restrict the codon-anticodon interaction of tRNAIII Arg.


[IR-spectroscopic study of cytochrome C-phospholipid lipoprotein and proteolipid model membranes].

It is shown by the methods of IR-spectroscopy and peptide hydrogen-deuterium exchange that a) considerable changes in the protein spectra occur (beta-conformation in the protein structure appears) during the interaction in water of cytochrome c molecules with lipid membranes containing negatively charged polar groups; b) further significant changes of the protein spectrum occur under the action of 1% OsO4 and heating up to n plus 95 degrees C; c) the conformational state of the pure protein in water; after the treatments of the proteolipid memebranes with 1% OsO4 and heating up to n degrees C no significant changes of protein spectrum occur, that may suggest hydrophobic interactions between the protein and lipids; d) the treatment of both pure cytichrome c and the model membranes with 1% glutaraldehyde, 30, 60% ethanol and acetone solutions in water does not reveal substantial changes in IR-spectra of the protein moiety.


[Reduction paths of reaction centers of photosystem I of photosynthesis].

The behaviour of ESR signal I in blue-green algae and chloroplasts is considered under various illumination and with various previous treatment. It is shown that the main process determining the kinetics of P700 plus reduction is the reaction with reduced components of the electron transfer chain between the two photosystems. If the accumulation of the products is inhibited by any way, then the reduction rate depends on the reactions with reducing agents in the incubation medium accumulated under irradiation before experiment. These exogenic reducing agents may be removed from the chloroplasts with repeated washings, and from algae-by means of the dark incubation for a long time. The relation of cyclic and noncyclic electron transfer is considered with and without artificial cyclic flow mediator.


The mechanism of ferrocytochrome C oxidation by a horseradish isoperoxidase.

The oxidation of ferrocytochrome c catalysed by highly purified horse-radish isoperoxidase P2 was studied kinetically. To take into account the low turnover number of the enzyme and the tendency to autocatalytic oxidation of ferrocytochrome c, experimental conditions were used which prevented us from using the steady-state treatment. According to kinetic results reported by several authors, a kinetic scheme involving a ternary complex between the enzyme and the substrates was postulated and simulated on a hybrid computer. By assuming that the interaction of peroxidase with hydrogen peroxide is much faster than the interaction with ferrocytochrome c, one can verify that this scheme explains the fact that initial velocity does not vary in relation to the hydrogen peroxide concentration and that a sudden change of slope occurs in the kinetic curve for an initial hydrogen peroxide/ferrocytochrome c ratio lower than 0.5.


[Oscillator generator in the lower portion of the glycolytic system].

A mathematical model explaining the mechanism of autooscillation generation in the lower part of the glycolytic system is suggested. The model is based on substrate inhibition of glyceraldehydephosphatedehydrigenase (GAPDH) with glyceraldehydephosphate (GAP). Pyrimidinenucleotides (NADplus and NADH) are shown not to play an important role in the given oscillation mechanism and can not be considered to simplify the model.


On the control of septation in Escherichia coli.

Mutants of E. coli defective in cell septation (ftsA to ftsG, conditional thermosensitive mutants isolated by Ricard and Hirota) were studied with respect to their membrane protein composition, murein hydrolase activities and rates of synthesis of murein and phospholipids. Three classes of mutants have been distinguished: 1) those affected in both murein and phospholipid synthesis; 2) those affected in either murein or phospholipid synthesis and 3) those affected in neither of these parameters. Overall murein hydrolase activities, after activation, is of the same order in all the mutants screened. In addition to soluble products of murein splitting, we have found insoluble products that appear to be in dynamic equilibrium with the murein of the sacculus. Endogenous levels of cyclic adenosine 3',5'-monophosphate measured after blocking septation showed no variation. This suggests that the cyclic nucleotide is not involved in the metabolic control of septation.


Effects of microtubule inhibitors and cytochalasin B on thyroid metabolism in vitro.

Mitotic spindle inhibitors (colchicine, vinblastine, vincristine, 020, ethanol) and cytochalasin B inhibit the phagocytosis of colloid by thyroid cells and the secretion of thyroid hormones. This inhibition has been linked to interferences with the microtubular microfilament system of the follicular cell. In order to test the possibility of using such inhibitors to selectively block secretion, the action of suppressing or highly inhibitory concentrations on other metabolic parameters has been studied on dog thyroid slices in vitro: glucose oxidation, lactate formation, iodide binding to protein, cyclic 3'5' AMP accumulation. It is shown that at a concentration of 10 mM colchicine is entirely non specific as it greatly inhibits all facets of metabolism and all the stimulatory effects of cyclic 3'5' AMP and thyrotropin. The other mictrotubule inhibitors, although affecting thyroid metabolism in various ways were more specified. The enhancement by vineblastine of glucose oxidation ald iodine binding to proteins suggests an activation of they thyroid H2O2 generating system. D2O on the other hand selectively inhibits secretion and the binding of iodide to proteins. Cytochalasin B, presumably by inhibiting hexose transport, decreased glycolysis and the uptake of iodide. However this effect cannot account for the complete inhibition of thyroid secretion.


[The effect of SH group inhibitors on phosphate transport in Chlorella pyrenoidosa].

The effect of Sulphydryl reagents have been investigated. pCMB inhibits the transport of phosphate in Chlorella pyrenoidosa. This inhibition is immediate and does not change as a function of time of incubation. This inhibition affects non starved and starved cells (phosphate omitted). pCMPS and Mersalyl act in the same manner as pCMB. When these compounds are used at low concentrations, inhibition of phosphate uptake is observed only in starved cells. The substrate (phosphate) cannot provide protection against this inhibition. NEM inhibits phosphate uptake and this inhibition increases as a function of time of incubation. When the time of incubation is very short (about 15 seconds) the effects seems to be superficial and NEM reacts with SH groups involved in the transport system. When phosphate is present (for 15 seconds of incubation with NEM) the inhibition is less important than when phosphate is omitted. The substrate protects against NEM, but this protection disappears as the incubation with NEM is prolonged. NEM inhibits phosphate uptake in non starved and starved cells, however, it is observed that the inhibition is less important in starved cells than in non starved cells. The authors conclude that two kinds of SH groups might exist in the phosphate transport system, one reacting with pCMB and the other with NEM.


[Applications of electron paramagnetic resonance to the study of biological molecules].

In a first part, an elementary theoretical introduction to ESR is given. In a second part, selected examples illustrate the possibilities of this method. Studies of hemoproteins, flavoproteins, model complexes and phospholipid systems are described. This review is made for biochemists who have no experience in ESR.


Electron spin resonance study of melanin treated with reducing agents.

The electron spin resonances (ESR) of several native and modified melanins have been determined. Melanins isolated from black wool and synthesized from 3,4-dihydroxyl-L-phenylalanine (L-DOPA) and tyrosine all show similar ESR signals. Modification of the isolated melanins by treatment with reducing agents causes some lightening in color and slight changes in the ESR spectra. Lithium and liquid ammonia (Birch) reduction applied to melanins from wool and L-DOPA gave very different results, as reflected by ESR spectra, but in both cases the changes were much greater than those produced by other treatments. In general, reductive treatments in nonaqueous media in the presence of metals increase the free radical content and line width, whereas treatment in aqueous media resulted in decreased free radical content. These observations are consistent with a melanin pigment which is an irregular polymer and has unpaired electrons localized on different but similar monomer units.


Iontophoresis of cyclic AMP.

The design, calibration, and operation of a source of controlled amounts of cyclic AMP (c-AMP) are described. Typically, 1.5 s pulses containing 10(10)-10(-12) molecules of c-AMP can be delivered to a region about 10 mum in diameter on an agar plate. The resulting concentration profiles are given as functions of distance and time. The diffusion coefficient of c-AMP in agar was measured to be 0.97 times 10(-5) cm2-s-1 at 21 degrees C.


Stimulation of late interphase Dictyostelium discoideum amoebae with an external cyclic AMP signal.

The microelectrode system described in the accompanying paper was used to investigate properties of fields of Dictyostelium discoideum amoebae in late interphase. Cells in the fields were competent to respond chemotactically to, and to relay, a c-AMP signal, but not to produce an aggregative signal autonomously. The experimental results are generally consistent with c-AMP being the sole compound required for chemotaxis and signal relaying. A periodic signal from the microelectrode can initiate and control aggregation and can complete with spontaneously arising aggregates. The electrode was used to measure the refractory period for relaying which decreases from 9 min or more to between 2 and 3 min with increasing developmental age, and to measure thresholds for chemotaxis and signal relaying. The results are discussed in relation to models for the control of aggregation in D. discoideum.


The effects of protein deprivation on cell-mediated immunity.

The effect of protein insufficiency on immune responsiveness was investigated in inbred mice fed isocaloric diets low and normal in protein. Mice were tested for aberrations of cell-mediated and humoral immunities. Protein insufficiency enhances cell-mediated immunity while depressing or not affecting humoral immunity. Cell-mediated immunity was enhanced in testing: allogeneic skin graft rejection, spleen cell responsiveness to phytohemagglutinin, the graft-vs-host reaction, macrophage function, and resistance to viral and bacterial infections.


Immune deficiency in congenital rubella and other viral infections.

No immunologic explanation has been found for the chronicity commonly observed in congenital viral infections. In the presence of a humoral immune response, there is continued viral excretion--in rubella for many months, and in the herpes viruses perhaps for life. Infection with rubella early in utero has a profound effect on the developing immune system. Defects observed are: complete immune paralysis, PHA unresponsiveness, immunoglobulin abnormalities, and loss of antibody to rubella. These defects are transient; absence of IgA may be permanent. No such defects have been observed in other congenital viral infections, but precocious development of immune globulin levels and germinal follicles occurs.


The pituitary dwarf mouse: a model for study of endocrine immunodeficiency disease.

Pituitary dwarf mutants of the Snell-Bagg and Ames mouse strains develop severe immunodeficiency of the thymus-dependent system which frequently leads to a fatal wasting syndrome. This immunodeficiency is a consequence of defective pituitary influences which will cause 1) an inadequate production of immunocompetent cells due to a central developmental defect primarily affecting the thymus and 2) the inability of immunocompetent cells to undergo a rapid and efficient antigen-induced proliferation and differentiation into antibody-forming cells. The relevance of the dwarf mouse model to a possible association between human immunodeficiency and endocrine disease is briefly discussed.


The effects of ATP on platelets: evidence against the central role of released ADP in primary aggregation.

The influence of freshly purified ATP on the effects of aggregating agents on human platelets was studied. ATP inhibited aggregation induced by ADP competitively (Ki = 20 muM) and immediately without need for prior incubation. ATP had no effect on primary aggregation induced by adrenaline, thrombin, vasopressin, or 5-hydroxytryptamine (5HT). ATP inhibited the shape change and the consumption of metabolic ATP induced by ADP but did not inhibit these effects when induced by thrombin, vasopressin, or 5HT. ATP counteracted the inhibition by ADP of PGE1-stimulated cyclic AMP production in platelets but did not reduce inhibition by adrenaline. It is concluded that adrenaline, thrombin, 5HT, and vasopressin each can induce primary aggregation of human platelets by a mechanism independent of extracellular ADP.


A survey of intestinal parasitism in a community on the Pan American Highway route in eastern Panama.

A survey of intestinal parasitism was conducted at Yaviza, a town in eastern Panama's Province of Darien. Single stool specimens from 202 persons, representing 11 percent of the town's total population, were examined. In all, 181 (90 per cent) of the specimens examined were found positive for one or more potentially pathogenic parasites. The most common parasite found was Trichuris trichiura, which was observed in 80 per cent of the samples. Others were Ascaris lumbricoides, in 62 per cent of the samples; hookworm, in 41 per cent; Entamoeba histolytica, in 16 per cent; Stronhyloides stercoralis, in 7 per cent; Giardia lamblia, in 5 per cent; and Hymenolepis diminuta, in 0.5 per cent. The aim of the survey was to gather baseline data on parasite prevalence before the Pan American Highway passes through this area. Construction of the highway is producing marked environmental change. in eastern Panama, and it is anticipated that the date from the survey will prove useful in later efforts to assess the health impact of that change.


Investigation of the vasodilator and antisecretory role of prostaglandins in the rat gastric mucosa by use of non-steroidal anti-inflammatory drugs.

1 The effects of non-steroidal anti-inflammatory drugs on gastric acid secretion and mucosal blood flow were studied in the rat. 2 Indomethacin, in ulcerogenic doses, caused a dose-dependent rise in pentagastrin-stimulated acid secretion, but decreased mucosal blood flow per unit acid secretion. 3 During resting conditions, indomethacin had no significant effect on acid output, but reduced mucosal blood flow. 4 Pretreatment with indomethacin, phenylbutazone or meclofenamate potentiated the secretory response to dibutyryl cyclic adenosine 3',5'-monophosphate. 5 Indomethacin markedly reduced the mucosal prostaglandin-like activity at a time when mucosal erosion formation had reached steady levels. 6 These results provide evidence that prostaglandins have a local role in the regulation of blood flow and acid secretion in the rat gastric mucosa, and suggest that non-steroidal anti-inflammatory drugs cause mucosal erosions by disrupting these processes.


The effect of reserpine on hypothalamo-pituitary-adrenocortical function in the rat.

1. The effect of reserpine on hypothalamo-pituitary-adrenocortical (HPA) function in the rat was investigated by the use of direct and indirect indices of pituitary adrenocorticotrophic activity. 2. Administration of a single dose of the drug induced prolonged hypersecretion of corticotrophin (ACTH). 3. Corticotrophin release in response to the drug no longer occurred after repeated daily injections, indicating that some form of 'adaptation' occurred. 4. The increase in HPA activity normally caused by exposure to cold was prevented by reserpine once 'adaptation' to the drug had been produced. 5. Inhibition of stress-induced ACTH release was due neither to depletion of pituitary stores of the hormone, nor to a corticosteroid feedback effect.


The effects of adenosine triphosphate and adenosine diphosphate on transmission at the rat and frog neuromuscular junctions.

1 The effects of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were investigated on evoked end-plate potentials (e.p.ps) and on miniature end-plate potentials (min. e.p.ps) recorded from muscle fibres of the rat diaphragm and the frog sartorius.2 ATP and ADP decreased the quantum content of the e.p.ps and the frequency of the min. e.p.ps. The maximum effects produced by the two substances were similar.3 The potency of ATP was found to be similar to that of adenosine. In the presence of adenosine, in a concentration producing its maximum effect, the addition of ATP had no further effect. This is compatible with the idea that ATP acts in the same way as adenosine.


Hepatic resection.

One hundred and twenty-five hepatic resections were done in the University Surgical Unit at the Queen Mary Hospital, Hong Kong, from 1964 to 1974 for conditions varying from primary hepatoma and recurrent pyogenic cholangitis to spontaneous rupture of the liver due to a bleeding haemangioma. The overall mortality was 17.6 per cent. A case was classed as an operative death unless the patient was discharged from hospital. There has been long term survival after resection for primary carcinoma of the liver and no mortality when resection was carried out for benign conditions.


Effect of surgical trauma on plasma concentrations of cyclic AMP and cortisol.

The response of plasma cortisol and cyclic AMP concentrations to surgical trauma was examined in 7 general surgical patients. Five patients made uncomplicated recoveries and showed peak cortisol levels at 6 hours postoperatively, returning to normal by 24 hours. On patient developed postoperative pneumonia and the plasma cortisol remained elevated up to 48 hours, and the final patient showed no cortisol response at all. The plasma cyclic AMP concentrations showed a consistent rise during the operation itself, which was statistically significant when compared with preoperative levels (P  less than 0.0125). Concentrations had fallen to normal by 6 hours in all the cases, except in the patient who developed pneumonia, where the plasma cyclic AMP also showed a prolonged elevation. The significance of these findings requires further elucidation, but the temporal relationship of the cyclic AMP and cortisol peaks would support the hypothesis that cyclic AMP may play a part in the mediation of ACTH release following surgical trauma.


Plasma corticotrophin levels in addison-schilder's disease.

Raised plasma immunoreactive corticotrophin (ACTH) levels were found in five boys with the sex-linked disorder progressive leucodystrophy associated with adrenal insufficiency (Addison-Schilder's disease) and in a symptom-free brother of one of them. Similar ACTH concentrations were found using two antisera, one against the N-terminal part of the ACTH molecule and the other against the C-terminal part. In one patient the circulating ACTH had normal biological activity as measured using the cytochemical ACTH bioassay. Immunoreactive beta/-melanocyte-stimulating hormone was also determined in one patient and found to be raised.


Biochemical studies on myelin isolated from the brains of patients with Down's syndrome.

Amyelination has been deduced from the data on chemical studies of myelin isolated from the brains of Down's syndrome. The lack of cholesterol and much reduced phosphohydrolase activity in mongol myelin possibly suggest a fault in the structure of myelin.


Raphe origin of serotonergic nerves terminating in the cerebral ventricles.

The dorsal and median raphe nuclei of the midbrain are known to contain the perikarya of origin of the major serotonergic (indoleamine) neurons projecting to the parenchyma of the forebrain. Lesions were placed in these nuclei to determine whether serotonin-containing nerve terminals in the cerebral ventricular system are also derived from the raphe nuclei. Brain tissue from control rats and rats 2-7 days after placement of raphe lesions was examined by fluorescence and electron microscopy. By the third day after lesion there was a marked reduction in the formaldehyde-induced fluorescence of supra-ependymal terminals. By the same time virtually all supra-ependymal terminals showed advanced degenerative changes as visualized by electron microscopy. There was a degeneration of supra-ependymal terminals in all parts of the cerebral ventricular system examined, including the epithalamic region (e.g., habenula and pineal recess; serotonin-containing terminals in the latter areas had previously been thought to arise from modified pinealocytes in the pineal recess). We conclude that most, if not all, supra-ependymal nerve terminals are derived from serotonergic cells of origin in the raphe nuclei.


Long-lasting facilitation of a synaptic potential following tetanization in the in vitro hippocampal slice.

Field potentials evoked by stimulation of afferent fibers in stratum radiatum were recorded in the CA1 region of the hippocampal slice maintained in vitro. Stimulation rates of 3-50/sec produced a large increase in amplitude of the population spike in CA1. This increase was maintained for several hours after the tetanization. The facilitation phenomenon appeared to be specific to the synapse of stratum radiatum afferents onto CA1 pyramidal cells since: (1) stimulation outside the radiatum layer did not produce the effect, (2) antidromic field potentials recorded in CA3 were unchanged, (3) EPSP threshold in CA1 was unchanged, and (4) alveus tetanization did not produce a facilitatory effect.


Subcellular analysis of the accumulation of estrogen by the brain of male and female rats.

Three experiments were preformed to provide additional information on the interaction of estrogen with subcellular components of the brain of male and female rats. In experiment 1 tritiated estradiol was administered to adult gonadectomized male and female rats which were then sacrificed 15,60 or 120 min later. Hypothalamic, cortical and pituitary samples were taken and were separated into nuclear and cytosol fractions. For the hypothalamic tissue from females nuclear concentration of radioactivity increased throughout the 2 h period while for males nuclear concentration rose during the first h and then declined. There was a significant sex difference in hypothalamic nuclear concentration of estrogen, male levels being lower. For both sexes cytosol levels progressively declined. For cortical tissue, nuclear radioactivity levels were low and relatively constant for both sexes, while cytosol levels fell during the 2 h period. Pituitary tissue showed a pattern in both nuclear and cytosol fractions which resembled the hypothalamic pattern although absolute levels were higher in the nuclear fraction. In experiment 2 male and female rats were administered labeled and unlabeled estradiol concurrently and were sacrificed 60 and 120 min later. Radioactivity levels were reduced in hypothalamic and pituitarynuclei, but not in cortical nuclei in comparison with animals not administered unlabeled hormone. In experiment 3 males and females were administered tritiated estradiol and were sacrificed 2 h later. The brain of each animal was split longitudinally. One half of each hypothalamic and cortical sample was subjected to nuclear separation while the other half was digested in tissue solubilizer before radioactivity counting. The former procedure showed a substantially greater nuclear concentration of radioactivity for hypothalamic tissue from females than from males. The whole tissue analysis showed only a slight sex difference for hypothalamic tissue. Sex differences were small by either procedure for cortical tissue. It was concluded that the hypothalamus of both male and female rats contains a limited capacit nuclear binding system for estrogen, but that the system is quantitatively less effective in binding estrogen in males.


Physiological and pharmacological evidence for a serotonergic projection to the hippocampus.

Rat hippocampal pyramidal cells were studied for their response to serotonin applied iontophoretically and to stimulation of the midbrain raphe nuclei. Ninety-two percent of the cells studied were inhibited by serotonin. Fourty-eight percent of the cells responded by inhibition to dorsal and median raphe stimulation. The inhibitory response to raphe stimulation was absent when the rats were pretreated with p-chlorophenylalanine (PCPA), a serotonin synthesis inhibitor; PCPA effects were alleviated by 5-HTP or 5-HT administration. The response to raphe stimulation was blocked by emthysergide and cyproheptadine. The responses to raphe stimulation were potentiated by chlorimipramine, a serotonin reuptake blocker. These data satisfy several of the criteria required to identify serotonin as the inhibitory neurotransmitter for the raphe-hippocampal pathway


Denervation in the primary olfactory pathway of mice. II. Effects on carnosine and other amine compounds.

Carnosine (beta-alanyl-histidine) is present in the olfactory bulb and olfactory eqithelium of mice and rats at 1-2 nmole/mg tissue. Peripheral deafferentation or central denervation causes a rapid, selective decrease of this depeptide from the reciprocal portion of the primary olfactory pathway. These data demonstrate the localization of carnosine within the primary olfactory chemoreceptor neurons and suggest a possible role for this compound in neural transmission.


A possible non-oncogenic effect of C type bone cell virus on serum calcium levels of potentially leukemic mice.

In bone of C3H/Fg mice, particles structurally identical to C-type leukemia virus arise from membranes of osteocytes and osteoblasts. Although these virus apparently do not induce morphologic or neoplastic change in bone they may have other, more subtle, effects. Thus, comparison of sera from male C3H/Fg mice, a high leukemia-prone strain, with C57BL and C3H/HeJ mice, low leukemia strains which do not contain C-type virus in bone, reveals that serum calcium levels are significantly lower in the former than in the latter. Further, when C3H/Fg mice develop frank leukemia there is a corresponding increase in virus particles while the serum calcium concentration levels fall to even lower values. The presence of leukemia itself appears not to be the cause as indicated by the failure of implanted lymphocytic leukemic cells in C3H/Fg mice to significantly affect serum calcium concentration. It is postulated that the effects of the virus could be due either to increased osteo blastic activity or to inhibition of osteocytic osteolytic activity or to both.


Comparison of whole calvarial bones and long bones during early growth in rats. Histology and collagen composition.

The distribution of ossified collagen (bone) and uncalcified collagen (fibrous tissue and cartilage) was compared histologically for rat and dog calvaria at birth. The relative amount of bone and uncalcified collagen was quantitated morphologically for rat calvaria during the first four weeks of rapid growth. Whereas dog calvaria are essentially ossified at birth, rat calvaria at birth consist mostly of fibrous tissue but rapidly become ossified with growth. Bacterial collagenase was used to separate uncalcified collagen from calcified collagen of whole membranous bones (frontal and parietal) and long bones (femur and humerus) at birth from man, monkey, dog, guinea pig, rabbit and rat. By this means quantitative changes in the relative fractions of the two forms of collagen were determined during the first eight weeks of postnatal growth for each type of rat bone. Quantitative biochemical data on whole rat bones (calvarium, femur, humerus) confirmed measurements based on histology which showed that at birth rat calvaria are mostly uncalcified as compared to other species whose bones are mostly ossified at birth. With growth rat membranous bones ossify more rapidly than long bones.


The effect of a myelin apoprotein on lipid structure: a spin probe study.

Spin probes, stable free radical derivatives of stearic acid and cholestanone, were used to observe the effects of the "Folch-Lees" protein isolated from the white matter of bovine brain on the organization and motion of lipid molecules. The incorporation of the organic solvent soluble form of this protein decreased the tendency of a variety of lipid molecules with zero, positive or negative net charges to arrange themselves close to the normal to the lipid bilayer. The aqueous form of the protein also had a profound chaotropic effect on the molecular geometry of the lipid, but only if the lipids had a net negative charge (the protein has a net positive charge in the pH range investigated). Examination of the ESR spectra indicated that this protein altered the geometry of the lipid structure without causing major changes in the mobility of the individual lipid molecules.


In vitro synthesis of estrogen glucuronides and sulfates by human renal tissue.

17beta-[6,7-3H]Estradiol (E2) was incubated with slices and homogenates of adult human renal tissue. The metabolites formed were identified by chromatography on DEAE-Sephadex, thin layer chromatography and crystallization with carrier steroids or steroid derivatives. The major metabolites formed by slices were estradiol-17-glucuronide (E217G), estrone sulfate and estradiol-3-sulfate. This is the first report of in vitro synthesis of estrogen sulfates by adult renal tissue. Minor quantities of the 3-glucuronides of estrone and estradiol were also found. An oxygen atmosphere appeared to stimulate the production of E217G. A time study with tissue slices showed similarities between the in vitro pattern of glucuronide synthesis and the excretion pattern of these compounds seen in earlier in vivo studies. Homogenates fortified with uridine diphosphoglucuronic acid formed the same pattern of glucuronide products but in lesser amounts. No sulfates were formed under these conditions. Testosterone did not act as a substrate in the experimental conditions used.


Biosynthesis of mitochondrial phospholipids using endogenously generated diglycerides.

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [gamma32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.


Adenovirus type 19 keratoconjunctivitis in Canada.

We describe an outbreak of epidemic keratoconjunctivitis occurring in Montreal during the winter of 1974. Adenovirus type 19 was the only virus isolated. We confirm the presence of type 19 adenovirus in Canada; it produces severe keratoconjunctivitis. The incubation period, method of spread and clinical findings resemble those seen in outbreaks of type 8 EKC. The prevalence of adenovirus type 19 in the population of Canada is unknown. Although some object to the use of the term EKC for infection caused by adenoviruses other than type 8, we believe that EKC should be regarded as an entity requiring virus isolation and antibody determination to identify the adenovirus type responsible for it.


Effect of immunization with attenuated Mycobacterium bovis (BCG) on experimental herpetic keratitis.

Mycobacterium bovis (BCG, Pasteur strain) was injected intraperitoneally into guinea pigs and rabbits on the 19th and 5th days prior to the intracorneal inoculation of Herpesvirus hominis. During a three-day and nine-day observation period, respectively, the BCG had no significant effect on the clinical course of the herpetic keratitis and no effect on the titer of virus present in the infected corneas.


The effect of caesium on adrenergic transmission in rabbit vas deferens.

The effect of caesium on the responses of rabbit vas deferens to transmural stimulation was investigated. The tissue responded to transmural stimulation with a phasic spike contraction followed bya sustained contractile response. The sustained response was inhibited by phentolamine and guanethidine and thus apparently results from noradrenaline release from adrenergic nerves. Addition of 2-5mM Cs+ greatly potentiated this secondary response without altering the sensitivity of the tissue to added (minus)-noradrenaline. This potentiation was not due to Cs+ decreasing the neuronal uptake of noradrenaline, or by Cs+ altering prostaglandin synthesis. Addition of 2mM Cs+ significantly increased the amount of (plus or minus)-[3-H] metaraminol released from tissues in response to transmural stimulation (5 Hz). It is suggested that caesium potentiated responses of rabbit vas deferens to transmural stimulation by increasing the amount of transmitter released per nerve impulse, possibly as a result of prolongation of the action potential.


Feedback inhibition of adrenocorticotropin release by corticosterone infusions in the adrenalectomized rat.

Advantage was taken of a specific and sensitive bioassay for rat plasma adrenocorticotropin (ACTH) based on the dispersion of rat adrenal cells with trysin, to investigate the relationship between plasma corticosterone concentration and inhibition of ACTH release under steady-state conditions achieved by graded rates (0-5.12 mug/min per 100 g body weight) of intravenous infusion of the steroid for 45 min in 28-day adrenalectomized rats. In contrast to prior reports involving suppression of stress-induced ACTH release, the inhibitory effect of corticosterone was shown, under our experimental conditions, to be exerted also on the basal rate of ACTH secretion. Indeed, a slight though not significant decrease of plasma ACTH concentration was observed with the corticosterone infusion rate of 0.64 mg/min per 100 g body weight, and further progressive and highly significant drops in concentration were recorded for infusion rates of 2.56 and 5.12 mg/min per 100 g body weight. An increase of the metabolic clearance rate of corticosterone, observed as a function of the infusion rate, was ascribed to saturation by the steroid of the plasma transcortin binding sites.


Phosphorylation of the membrane components of chromaffin granules: synthesis of diphosphatidylinositol and presence of phosphatidylinositol kinase in granule membranes.

Adenosine triphosphate (ATP) induces the release of catecholamines, endogenous ATP, and soluble protein from chromaffin granules isolated from the adrenal medulla. When ATP exerts this action, it is hydrolyzed by enzymes present in the granule membrane, and part of the Pi liberated from ATP is transferred to the protein and lipid of the granule membrane. The phosphorylated lipid component, which was identified by thin-layer and ion-exchange chromatography as diphosphatidylinositol, was formed from ATP and monophosphatidylinositol. This latter phospholipid was the substrate for the enzyme phosphatidylinositol kinase. Both substrate and enzyme are components of the granule membranes, because they have a similar subcellular distribution as dopamine beta-hydroxylase (a granule membrane marker). The formation of diphosphatidylinositol was Mg(2 plus)-dependent, it was further stimulated by Mn(2 plus), it was inhibited by N-ethylmaleimide and the reaction had an optimal pH of 5. The synthesis of diphosphatidylinositol was also shown to occur in chromaffin granules "in situ". during the stimulation of the adrenal medulla by acetylcholine.


The demonstration of transmissible gastroenteritis viral antigens by immunoelectrophoresis and counterimmunoelectrophoresis.

Immunoelectrophoresis of alkaline intestinal extracts, or the supernatants after precipitation of these extracts with 60% ammonium sulfate, prepared from piglets experimentally infected with the DL or Purdue strains of transmissible gastroenteritis virus, revealed up to three antigens. Two antigens migrated towards the anode, and the third migrated towards the cathode. Antigens with anodal or cathodal mobility were also demonstrated in the same materials by counterimmunoelectrophoresis, and the procedure for counterimmunoelectrophoresis was modified to detect both antigens in a single test.


Virus survival on inanimate surfaces.

The persistence of several types of viruses on hard, inanimate surfaces under different relative humidities, temperatures, and types of surfaces was investigated. No differences in survival on glass, vinyl asbestos tile, ceramic tile, and stainless steel were found. Under conditions of low humidity and room temperature, adenovirus, poliovirus, and herpes simplex virus survived for at least 8 weeks. Vaccinia and coxsackie viruses survived for at least 2 weeks but differences due to surfaces found in many environments, in addition to the laboratory, emphasizes the possible role of hard surfaces in the transmission of viruses.


Aerobic adaptation in yeast. II. Changes in enzyme profiles during a step-down anaerobic-aerobic transfer.

The levels of various enzymes and components of the glycolytic and respiratory pathways of the yeast Saccharomyces cerevisiae have been determined during a step-down, anaerobic-to-aerobic transition. These activities were determined as an adjunct to the respective metabolite data reported in the first paper in this series. It is clear from the data that anaerobic conditions induce an environment conducive to express glycolytic enzyme activities, while manifesting a differential induction/repression effect on oxidative enzymes. An NAD/NADH mediated mechanism is proposed to explain this difference. Of the enzymes assayed only cytochrome c oxidase shows any direct response to oxygen challenge and consequently it is suggested that the assembly of this enzyme is the trigger mechanism and rate-limiting step in aerobic adaptation.


Aerobic adaptation in yeast, IV. Alterations in enzyme synthesis during anaerobic-aerobic transitions in exponentially growing cultures.

As a corollary to the metabolite data obtained from yeast cultures undergoing an exponential anaerobic-aerobic phase transition, levels of various glycolytic and citric acid cycle enzyme activities have been monitored in these cells. The relation of the changes in these enzyme activities in cells grown on either glucose or galactose is discussed on light of different metabolic postures these cells demonstrate as a result of their transitions. A general discussion is presented which compares the results obtained in this series of papers from both step-down and exponential transfer experiments and relates these data to control of mitochondriogenesis in yeast.


Ultrastructure of the Alnus crispa var. mollis Fern. root nodule endophyte.

Nitrogen-fixing, field-obtained root nodules of the silky green alder were studied by transmission electron microscopy.  The nodule endophyte exhibited a prokaryotic cytology and was present in two forms: the hypha(0.3-1.0mum), which was branched and septate, and the vesicle (3-5mum), which was also septate and developed at the parental hypha tip.  Bacteria-like cells, previously observed in light microscopy studies, were not seen in the present work.  The actinomycete-like endophyte penetrated through the host cell wall and becane enveloped by a capsular material (0.1mum), the whole being enclosed by host membranes.  In some host cells, the endophyte appeared to lyse and become a mass of shrunken debris.  The fine structure of the Alnus crispa var. mollis root nodule endophyte was found to be similar to that of other nonleguminous root nodule endophytes.


An immunoprecipitin study of the incidence of influenza A antibodies in animal sera in the Ottawa area.

A survey of over 600 'normal' sera from 14 animal species by immunoprecipitin tests in cellulose acetate using viron antigens revealed a high incidence of precipitating activity against a broad range of influenza A virus strains, particularly A2hHong Kong/1/68 and /PR8. However, serum treatments trypsin-heat-periodate, NaIO4, V. cholerae receptor-destroying enzyme (RDE), or kaolin eliminated most precipitating activity, which suggests that it was due to "non-specific" inhibitors of influenze viruses. A resistant minority could not be identified as inhibitor or antibody on this basis. Precipitation of the influenza A major type-specific antigen in virus-soluble antigens by human 7S gamma globulin antibody (IgG), demonstrated to be specific for influenza virus, was established as a reference reaction to identify similar immunoprecipitin reactions occurring between virus-soluble antigens and normal or immune sera. Complement fixation tests provided supplementary evidence for the presence of influenza A antibodies in these sera. Influenza A antibodies were found in only a few sera of six animal species: cat, dog, rabbit, goat, chipmunk, and sheep. Thus the animal species examined in the Ottawa area have not revealed an unequivocal reservoir for human influenza A viruses.


Patterns of motoneuron dysfunction and recovery.

Electrophysiological studies have been carried out on five patients with neuropathies of different etiologies. In each patient serial estimates were made of the numbers of functioning motor units in various muscles. It was found that the intensity of the neuropathic process and the rate of recovery differed in a consistent way among the motoneuron pools investigated. The lesion was more severe in extensor digitorum brevis neurons than in thenar neurons, while the hypothenar ones were least affected. A stage of partial synaptic failure has been recognized in which a motoneuron appears to be no longer able to excite a muscle fiber, but still capable of maintaining certain trophic activities. By comparing the number of functioning motor units with the size of the maximum evoked muscle response it has been possible to detect the adoption of denervated muscle fibers by axonal sprouts from 'healthy' surviving neurons (collateral reinnervation). Lastly, in some muscles it appears that the adopted muscle fibers may subsequently be recaptured by the original motoneurons following recovery of the latter from the neurotoxic insult.


Clinical usefulness of gastric acidity studies.

Tests of gastric secretion are seldom diagnostic in the investigation of gastric and duodenal ulceration, but they can provide evidence that is helpful in arriving at a diagnosis. Common tests include basal acid output, pentagastrin-stimulated maximal and peak acid output, and the Hollander test. There is some evidence that they may be useful in selecting the type of operation for peptic ulceration. Indications for performing these tests relate to the Zollinger-Ellison syndrome, duodenal ulcer, recurrent dyspepsia after operation for duodenal ulcer, and decisions concerning the choice of operation for peptic ulcer. In order to perform these functions they must be properly conducted and interpreted; they are simply an adjunct to clinical judgement, and complementary to other laboratory tests.


Unilateral hypoglossal nerve atrophy as a late complication of radiation therapy ofhead and neck carcinoma: a report of four cases and a review of the literature on peripheral and cranial nerve damages after radiation therapy.

The case histories of four patients who developed hemiatrophy of the tongue from 3 to9 years after a course of curative radiation therapy for carcinomas of the head and neck are presented.  These patients subsequently followed for from 1 1/2 to 6 years withoutlocal recurrence of the tumor, distant metastasis, or involvement of other cranial nerves, indicative of only a unilateral hypoglossal nerve atrophy.  A review of the literatureshowed that peripheral and cranial nerve damages after radiation therapy have been reported for the optic nerve, hypoglossal nerve, oculomotor nerve, abducens nerve, recurrent laryngeal nerve, brachial plexus nerves and peripheral nerves of the extremeties.  Reviewof clinical and experimental data indicated that in most cases, the damages were probably caused by extensive connective tissue fibrosis around and infiltrating the nerve trunks.  Three possible types of peripheral and cranial nerve damages after radiation therapy are identified.


Complete and apparently specif local tumor regression using syngeneic or xenogeneic "tumor-immune" RNA extracts.

Syngeneic and xenogeneic RNA-rich extracts of lymphoid tissues were used in an immunotherapeutic regimen to treat strain 2 guinea pigs that were given intradermal injections of a uniformly lethal dose (1 x 10(6)) of line 10 diethylnitrosamine-induced transplantable hepatoma cells. When 1 X 10(7) syngeneic nonsensitive peritoneal exudate cells, 2.5 mg RNA from line 10-immune strain 2 guinea pigs or line 10-immune Rhesus monkeys, and 1.0 mg of a line-10 tumor-specific antigen preparation were injected s.c. under the tumor cells injected 5 days previously, complete local tumor regression in all treated animals was observed. If either nonsensitive peritoneal exudate cells, RNA, or line 10 tumor-specific antigen was omitted, or if Escherichia coli RNA or RNA from animals sensitized to a different tumor (line 1) was used, little or no tumor regression was observed, suggesting that the action of the RNA may have resulted in an antitumor response specific for the noplasm being treated. The long-term tumor-free survival of all treated animals indicates that the action of the RNA is systemic, since metastases are known to occur frequently by the time our therapeutic regimen was given. Also, in testing the biological activity of the "tumor-immune" RNA in the in vitro cell-migration-inhibition assay, both the syngeneic and xenogeneic RNA extracts could transfer tumor-specific immunological sensitivity, as demonstrated by the elaboration of migration-inhibitory factor by the RNA-treated nonsensitive peritonial exudate cells in the presence of the line 10 tumor-specific antigen.


Antitumor activity of selected amino acid derivatives against various tumor systems.

We have previously reported that from 350 amino acid (A-A) derivatives five were selected after the primary in vivo and in vitro screening tests. The five compounds which were found to possess potential antitumor activity against Ehrlich ascites carcinoma are as follows: beta-naphthalene-sulfonyl-DL-tryptophan (A-91), beta-naphthyl-aminomethyl-gamma-aminobutyric acid (A-144), N-ethylcarbaminomethyl-L-isoleucine (A-145), n-9-fluorenylactyl-L-phenylalanine (A-192), and N-propoinyl-L-valine (A-195). The effect on life prolongation and tumor growth of these selected A-A derivatives against various types of tumors, including ascites and solid tumors in mice and ascites hepatomas in rats, was examined. A-A derivatives were administered once daily 3 consecutive days starting 24 hours after tumor implantation. Experimental results showed that among the five A-A derivatives possessing considerable activity against Ehlich carcinoma, A-144 and A-145 were found to be more effective than chromomycin A and showed activity similar to that of cyclophosphamide against ascites Sarcoma 180. A-A derivatives showed slight antitumor activity against SR61 and L1210 leukemias. In rat ascites hepatoma, such as AH13, AH7974, AH60C, and Yoshida sarcoma, only A-145 showed a significant prolongation of the lifespan in the control groups. The five selected A-A derivatives significantly inhibited the growth of Nakahara-Fukuoka sarcoma and solid Sarcoma 180. These findings indicate that among the five A-A derivatives, A-15 appeared to be the most active against ascites and solid tumors.