Isolation of a product from the trypsin-digested glycoprotein of sciatic nerve myelin.

When purified rabbit sciatic nerve myelin, whether lyophilized or not, is treated with low amounts of trypsin (25 microgram/ml) for 0.5, 3, or 24 h the resulting protein patterns viewed on sodium dodecyl sulfate (SDS) gel electrophoresis are similar. The most striking feature of the trypsinized myelin is the accumulation of a heavy band at the basic protein position, molecular weight 19 000, which is accounted for as a degradation product of the PO protein, referred to as the TPO protein. The PO protein, the major glycoprotein of sciatic nerve myelin, as well as the 23K and P2 proteins and albumin, an absorbed component, are all partially degraded; most high molecular weight bands are lost. The TPO protein, isolated by gel filtration in 2% SDS on an agarose column, like the PO protein, is highly insoluble in aqueous solvents. It is a glycoprotein (8% carbohydrate), staining with periodic acid-Schiff reagent; containing 3  mannose, 1 galactose, 3 N-acetylglucosamine, 1 sialic acid, and 1 fucose residues and is identical to the nonasaccharide of the parent PO protein. The amino acid composition of the TPO protein, is similar to the PO protein, but has a much higher content of hydrophobic residues and begins with NH2-methionine. This suggests that the PO protein is an amphipathic membrane protein in which its more polar character is confined to the first third of its NH2-terminus. This polar domain is probably positioned above the lipid leaflet where it is accessible to trypsin which cleaves a sensitive lysinyl (or argininyl)-methionine linkage. The more hydrophobic domain (the TPO protein) is buried in the myelin bilayer where it is protected from further tryptic attack. Thus trypsin can serve as a useful probe of myelin structure.


Interaction studies of the 165 000 dalton protein component of the M-line with the S2 subfragment of myosin.

The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Sodium dodecyl sulphate electrophoresis revealed that the protein was homogeneous. Circular dichroism measurements indicated that the protein interacts with myosin and heavy meromyosin subfragment 2 (S2). There was an increase in negative ellipticity at 221 nm upon interaction, relative to the calculated values assuming no interprotein interaction. The net increaes in negative ellipticity at 221 nm as a result of interaction of M-protein with myosin and subfragment 2 were 600 degrees and 800 degrees respectively. When the protein was mixed with subfragment 2 in a 1 : 1 mol ratio in 0.5 M KCl/25 mM Tris buffer at pH 8.0, low speed sedimentation equilibrium studies gave a molecular weight of 235 000 +/- 10 000 for the complex, indicative of an interaction of the two components. On a Bio gel A 0.5 m column, M-protein and S2 when applied in 1 : 1 mol ratio, were eluted as a single symmetrical peak and a molecular weight of 230 000 was obtained for the complex from the observed elution volume. Both circular dichroism and sedimentation equilibrium studies indicated no interaction of M-line protein with light meromyosin and subfragment 1. Interaction of the 165 000 component with the flexible hinge region of myosin may have special significance in terms of the mechanism accounting for the reversible expansion of the interfilament distance which occurs during contraction.


Proteolytic fragments from the lobster myosin molecule.

The fragments produced by proteolysis of lobster abdominal muscle myosin with trypsin, alpha-chymotrypsin and papain have been investigated by sodium dodecyl sulfate (SDS) gel electrophoresis. Essentially monodisperse populations of long rods are produced by alpha-chymotryptic and papain digestion of rabbit myosin but corresponding digestion of lobster myosin yields multicomponent species. Similarly the low ionic strength insoluble fraction from tryptic digestion of lobster myosin is polydisperse in contrast to essentially monodisperse light meromyosin from rabbit myosin. Comparative tryptic digestion of rabbit and lobster myosin papain long rods shows that the latter have five susceptible cleavage sites in the subfragment-2 region while rabbit long rods have only one: both long rods appear to have three cleavage sites in the light meromyosin region. The fragments produced by tryptic digestion of rabbit myosin papain long rods have been tentatively identified by comparison with fragments isolated from papain digests of rabbit heavy meromyosin and tryptic digests of rabbit light meromyosin. The results suggest differences in sensitivity to enzymic proteolysis between the subfragment-2 regions in rabbit and lobster myosin as well as relative differences in proteolytic sensitivity between the subfragment-2 and light meromyosin region within the individual molecules. Partial explanation of the observation is proposed on the basis of differences in heavy chain compositions.


Binding of galactose and lactose to ricin. Equilibrium studies.

The interaction of ricin, one of the two lectins of Ricinus sanguineus, with its specific ligands galactose and lactose (4-O-beta-D-galactopyranosyl-D-glucopyranose) has been studied by means of equilibrium dialysis, analytical ultracentrifugation and fluorescence polarization. In the studied concentration range, only one molecule of galactose is bound per molecule of ricin with an association constant, Ka = 6900 m-1 at 4 degrees C. Scatchard plots of equilibrium dialysis data show that two molecules of lactose bind to one molecule of ricin, without modification of molecular weight of the lectin. Together with results of microcalorimetric experiments and agglutination of erythrocytes by ricin, equilibrium dialysis data indicate that the lectin contains two distinct saccharide binding sites. Regardless of the existence of extended sites, it is not possible to select between the two models: (a) two independent sites (Ka1 = 35 000 M-1, Ka2 = 2800 M-1 at 4 degrees C) or (b) two identical sites with negative cooperativity.


Isolation and characterization of glycoproteins from canine tracheal mucus.

Three homogeneous glycoproteins were isolated from reduced and S-carboxy-methylated canine tracheal pouch mucus by gel filtration and ion-exchange chromatography. Initial fractionation was carried out on Sephadex G-200; chromatography of the excluded Sephadex G-200 fraction on Bio-Gel A-15 m yielded two high molecular weight glycoprotein fractions. Following rechromatography on the same column, the main fraction behaved as an electrophoretically homogeneous high molecular weight (581 600) glycoprotein, with a high carbohydrate content (80%) and a single amino-terminal amino acid (arginine). Ion-exchange chromatography (DEAE-cellulose) of the included Sephadex G-200 fraction yielded two electrophoretically homogeneous glycoproteins of lower molecular weight (20 800 and 24 600, respectively). A single amino-terminal amino acid, glycine and alanine, respectively, was detected for each glycoprotein. Chemical analysis of these three glycoproteins revealed the presence of fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid and sulfate monoester. The high molecular weight glycoprotein had a higher hexose, sialic acid and sulfate content, per mg of protein, than the low molecular weight glycoproteins. The results of the alkaline borohydride treatment indicated that the majority of the carbohydrate chains of these glycoproteins are linked to the protein core through O-glycosidic bonds involving N-acetylgalactosamine and serine or threonine.


Raman spectroscopy of intact feline corneal collagen.

The Raman Spectrum of Collagen is presented from feline corneas which were fresh and intact, heat denatured, and incubated in 2H20. Two bands in the amide I region at approx. 1630 cm-1 and approx. 1660 cm-1 and two bands at ca. 1270 cm-1 and 1247 cm-1 in the amide III region appear in the Raman spectrum of fresh and heat denatured corneal collagen. The two amide III bands have been assigned to amide III vibrations in the polar and non-polar regions of the protein. Only one small amide I band at approx. 1650 cm-1 appears when corneas are treated with 2H2O suggesting that some portion of the Raman peaks in the amide I region for corneas in water is associated with water vibrations. Feline corneal collagen fibrils do not appear to dissociate appreciably upon heating to 70 degrees C. In fact, heated corneas appear structurally similar to corneas aged 30 h at 50 degrees C. We suggest that the swelling induced by heating and aging is predominantly caused by water being absorbed and remaining between the collagen fibrils, causing a slightly more disordered collagen matrix.


Circular dichroism studies on the effects of sodium dodecyl sulfate on the conformation of some phytohemagglutinins.

Conformation and conformational transitions of phytohemagglutinins (lectins) from Arachis hypogaea (peanut), Lens culinaris (lentil), Glycine max (soybean), and Ricinus communis (castor bean) were studied by the circular dichroism probe. The lectins were found to possess a high amount of the pleated sheet (beta) conformation. The tertiary structure and main chain conformation of the lectins were disorganized by sodium dodecyl sulfate (SDS) and the main chain was reconstructed by relatively high concentrations of SDS (weight ratio of SDS to protein about 5-8 to 1) into new orders of higher helix content than in the native protein. More helix was formed in acid solutions than in nearly neutral solutions. The disorganization of the native tertiary structure by SDS was expressed in a decrease of the circular dichroism bands related to the tyrosine and tryptophan chromophores and in appearance of the vibronic fine structure in the phenylalanine band zone. The tertiary structure of the L. culinaris and R. communis lectins was more sensitive to SDS than the tertiary structure of the two other lectins.


Quantitative analysis of the spin equilibrium of cytochrome P-450 LM-2 fraction from rabbit liver microsomes.

The LM-2 fraction of cytochrome P-450 from rabbits in the presence and in the absence of substrate (benzphetamine) is shown to be a thermal mixture of a high spin (S = 5/2) and a low spin (S = 1/2) form each of which exhibiting its individual optical basic spectrum with the Soret maxima at 387 nm and 417 nm for the high spin form and the low spin form, respectively. The equilibrium constants and thermodynamic parameters describing the spin transition and the substrate binding have been evaluated from the temperature and substrate difference spectra, respectively. These two interacting equilibria are presented in terms of a thermodynamic model, which provides a clear quantitative description of the properties of the cytochrome P-450 substrate system. From the thermodynamic model also the cause of the substrate difference spectra can be explained. The importance of the spin shift in the presence of substrate with respect to the reduction rate is discussed.


Human spectrin. I. A classical light scattering study.

Human spectrin heterodimers were analyzed in solutions containing different amounts of salt employing the classical light scattering technique. 1. At 22 degrees C the radius of gyration of isolated human spectrin heterodimers in 0.1 M NaCl aqueous solution (pH 7.3) was found to be about 22 nm. 2. The radius of gyration of isolated human spectrin heterodimers was found to increase to about 40 nm as the ionic strength of the spectrin solution (pH 7.3) was reduced to about 1 mM. 3. The light scattering study indicates that the isolated human spectrin heterodimers were highly expanded and flexible molecules with a contour length exceeding about 140 nm.


Human spectrin. II. An electro-optic study.

The electrically induced birefringence of human spectrin heterodimer solutions has been studied. 1. Human spectrin heterodimers were found to have a specific Kerr constant Bsp = +(5 +/- 2) . 10(-11) m4/(V2 . kg). 2. Human spectrin exhibited a birefringence relaxation time tau decay = (2.0 +/- 0.3) microseconds. 3. The electro-optic study indicates that human spectrin heterodimers have a contour length of more than 40--50 nm.


Parvalbumins from coelacanth muscle. I. General survey.

Parvalbumins from coelacanth (Latimeria chalumnae) myogen have been isolated by gel filtration of Sephadex G-75 and DEAE-cellulose chromatography. Disc electrophoresis and cellulose acetate electrophoresis showed the homogeneity of the three first major parvalbumin peaks (pI = 5.44, pI = 4.95 and pI = 4.52). The fourth component was partially resolved into two more parvalbumins (pI = 3.78 and pI = 3.50) by preparative gel electrophoresis. Amino acid analyses and tryptic peptide maps separated the five components in two major categories. The two less acidic components differ only in the presence or absence of an N-terminal blocking group. The three more acidic components constitute the second category; in spite of this heterogeneity, they share the same amino acid sequence.


Thermodynamic studies of the interaction of alpha-chymotrypsin with water. II. Statistical analyses of the enthalpy-entropy compensation effect.

Differential enthalpies (deltaH) and entropies (deltaS) of the interaction of water with a high and low temperature conformer of alpha-chymotrypsin were determined previously by multitemperature sorption measurements. The changes in (deltaH) and (deltaS) with water content of the protein were found to show a pronounced compensation pattern. It is known that van 't Hoff data may exhibit enthalpy-entropy compensation, which is entirely due to statistical error propagation. To discriminate between artifactual and significant compensation, the experimental results are analyzed by statistical methods. The results of two different statistical analyses show that a linear, chemically caused compensation effect can be established for the interaction of water with both chymotrypsin conformers. The compensation temperature beta = deltaH/deltaS was found to be 433 +/- 22 K. The compensation effect is detectable only in the water content range above the monolayer volume (upsilonm), computed by the Brunauer, Emmett and Teller equation. This result is discussed in terms of a monolayer hydration mechanism, formulated on the basis of previous thermodynamic results: The interaction of the first water monolayer with the charged and polar surface area of the dry protein, largely stabilizes its tertiary structure. Further water addition then occurs to a practically invariable protein surface. According to this mechanism (which ensures a maximum of conformational stability with a minimum of hydration water), large conformational changes can be expected to occur mainly in the monolayer water content range. This expectation is confirmed by extra-thermodynamic data (infrared and X-ray measurements). The thermodynamic quantities of the sorption process are thus governed by conformational effects below upsilonm. Above the monolayer water content range, however, the water binding process per se strongly predominates. The deltaH/deltaS compensation effect established for this water content range, is thus attributable to phase transitions of water molecules from the gas (or liquid) phase to the protein-bound state (or vice versa). A possible relationship between the linear compensation effect established in this study, and the compensation phenomenon observed in reactions in aqueous solution is discussed.


Parvalbumins from coelacanth muscle. III. Amino acid sequence of the major component.

The primary structure of the major parvalbumin (pI = 4.52) from coelacanth muscle (Latimeria chalumnae) has been determined. Sequence analysis of the tryptic peptides, in some cases obtained with beta-trypsin, accounts for the total amino acid content of the protein. Chymotryptic peptides provide appropriate sequence overlaps, to complete the localization of the tryptic peptides. Examination of the amino acid sequence of this protein shows the typical structure of a beta-parvalbumin. Its position in the dendrogram of related calcium-binding proteins corresponds to that usually accepted for crossopterygians.


Hemoglobin Detroit: beta95 (FG2) lysine leads to asparagine.

Hb Detroit is a mutant which migrates between Hb A and Hb J Baltimore on cellulose acetate (pH 8.5), and with Hb A on citrate agar (pH 6.0). Globin chain analyses in alkaline and acid buffers reveal an abnormal beta chain with a mobility between the betaA and betaJ Baltimore chains. Structural characterization of this abnormal chain shows that lysine at position 95 is replaced by asparagine. No hematological abnormalities could be attributed to the presence of the mutant, and the oxygen affinity properties of the stripped hemoglobin are similar to those of Hb A. The beta95 residue which is substituted in Hb Detroit and also in Hb N Baltimore ((beta95 Lys leads to Glu) does not appear to be in a critical functional area of the molecule.


The partial amino acid sequences of the two H2B histones from sperm of the sea urchin Psammechinus miliaris.

Two new histone H2B variants have been isolated from sperm cells of the sea urchin Psammechinus miliaris. They have been designated sperm histone H2B(1) Psammechinus and sperm histone H2B(2) Psammechinus. Both histones are highly homologous to the previously described sperm histones from Parechinus angulosus (Strickland et al. (1977) Eur. J. Biochem. 77, 263--275 and 277--286). The amino acid sequences of the Ps. miliaris sperm histones, though highly homologous, are not identical to the amino acid sequence derived from the codon sequence of a histone H2B gene, characterized from the same organism by Birnstiel et al. ((1977) Nature 266, 603--607).


Primary structure of hemoglobin from trout (Salmo irideus). Amino acid sequence of alpha chain of Hb trout I.

The amino acid sequence of the alpha chain of the hemoglobin component of trout's blood which is devoid of heterotropyc phenomena, i.e. Hb trout I, is presented. The sequence has been determined by analyzing the soluble tryptic peptides obtained from the whole globin and the peptides obtained after redigesting the insoluble 'core' with chymotrypsin. Alignment of the peptides with the structure of human as well as carp and Catostomus clarkii alpha chains shows that Hb trout I alpha chain differs from the corresponding human protein by 43% amino acid substitutions and from the two other fish by 34.5% and 33.1%, respectively. Further comparison of sequence data available for the N-terminal region suggests that the divergence between Hb trout I and IV, the other major hemoglobin component of trout's blood, is greater than that found between each trout hemoglobin and the other two fishes (carp and C. clarkii.).


The shape of spectrin molecules from human erythrocyte membranes.

Purified spectrin dimers and tetramers have been directly visualized by low-angle shadowing. The 9-S heterodimer is an asymmetric flexible molecule about 1000 A in length, its constituent monomer polypeptides forming two strands which in many molecules are individually visible, lying partially separated from one another or twisting round each other in a loose double helix. The 12-S tetramer is formed by the end-to-end association of two heterodimers, without overlap. The protein bears no physical resemblance to myosin.


Formation of the two-dimensional hexagonal lattice of bacteriorhodopsin in reconstituted brown membrane.

The aggregation state of reconstituted bacteriorhodopsin molecules in the brown membrane has been investigated by X-ray diffraction and CD spectra. It has been confirmed that reconstituted bacteriorhodopsin molecules form the hexagonal lattice spontaneously whereas bacterioopsin molecules do not.


Disulphide bridging of calf thymus histone H3 by 5,5'-dithiobis(2-nitrobenzoic acid).

Electrophoretic analysis of the reaction products of calf thymus histone H3 with 5,5'-dithiobis(2-nitrobenzoic acid), under mild conditions, shows that different oxidized forms of this histone are originated by disulphide interchange. One of the species detected is the intramolecular oxidized monomer of histone H3.


Isolectins of Phaseolus vulgaris. Physicochemical studies.

By a combination of solubility fractionation, continuous free-flow high voltage electrophoresis, molecular sieve and affinity chromatography on fetuin-Sepharose 4B, several lectin fractions of different isoelectric point were obtained from the seeds of Phaseolus vulgaris cv. "Processor". The albumin isolectin range comprised of five major lectin components with isoelectric points between pH 4.6 and 5.2, while the partly overlapping globulin isolectins contained several more lectin components with higher isoelectric points. The two groups had similar amino acid and sugar composition but were only partially identical by immunochemical criteria. The isolectins were also shown to be similar by physicochemical measurements with a common protomer weight value of about 119000. Values of so20,w, 6.84 and 6.76 S, and of V av, 0.712 and 715 ml/g, for the albumin and the globulin lectins, respectively, also indicated a close similarity. Both groups contained, in a slow equilibrium with the protomer, just over 10% of dimer and oligomers. In addition, the albumin lectins also contained a small, 2 S, dissociation product in a slow equilibrium with the protomer. The hydrodynamic data obtained for the albumin isolectins: [eta] = 0.063 dl/g; beta = 2.42 . 10(6) and f/fo = 1.56 indicated a highly voluminous particle with an effective volume of 8.6 . 10(-18) ml. This type of molecular arrangement was general for the major P. vulgaris seed glycoproteins and was, thus, suggested to be of potential importance for their physiological functioning during seed maturation and germination.


Glucose in vertebrate skeletal muscle proteins.

Neutral sugar was found in proteins extracted with acid chloroform/methanol from skeletal muscle of frog, normal and dystrophic chicken as well as in conventional preparations of frog tropomyosin and rabbit myosin. Whenever tested, the sugar content remained unaffected by two extractions with 5% trichloroacetic acid and by treatment with acid (0.1 N hydrochloric acid) or alkali (0.1 N sodium hydroxide, 60--90 min, 100 degrees C). These and other chemical properties favor the notion that sugar is present in covalent linkage to protein rather than as an attending contaminant. In every protein studied sugar profiles consisted of glucose only except rabbit myosin where it was accompanied by another sugar, presumably ribose.


Studies on cephalopod rhodopsin. Fatty acid esters of sucrose as effective detergents.

Squid rhodopsin was extracted with solutions of fatty acid esters of sucrose (monolaurate and monostearate) and purified by DEAE-cellulose and concanavalin A-Sepharose affinity chromatography. The purified rhodopsin (A280/A480 = 2.5) contained 2.3 mol of glucosamine and 1.2 mol of phospholipid per mol of rhodopsin. The photoproduct metarhodopsin was also stable in these detergent solutions as in digitonin solution. Concanavalin A had no affinity for retinochrome.


Multiple forms of albumin and their conversion from pro-type to serum-type in rat liver in vivo.

Purified rat serum albumin was resolved into five forms by polyacrylamide gel isoelectric focusing, while albumin isolated from the subcellular fractions of rat liver consisted of those of serum albumin and four additional forms. This latter four forms were identified as proalbumin, since they were converted to those of serum albumin by limited proteolysis with trypsin. This method was applied to the determination of the conversion site of proalbumin in the liver, revealing that substantial conversion of proalbumin occurs in the Golgi cisternae as well as in the secretory vesicles.


Human pituitary thyrotropin. Isolation of alpha and beta subunits by hydrophobic interaction chromatography.

Various conditions for the dissociation of highly purified human pituitary thyrotropin into subunits have been investigated. Dissociation on a preparative scale was accomplished by treatment with 1 M propionic acid at 32 degrees C for 16 h. The isolation of one alpha and two beta subunits was achieved by hydrophobic interaction chromatography on pentyl-Sepharose-4B. Radioimmunological technique was utilized to classify the subunits in accordance with current nomenclature and also to express their activities. The activities of the subunits overlapped insignificantly (less than or equal to 0.3%) and their content of intact thyrotropin was negligible (less than 0.05%). The characterization of the subunits included determination of their amino acid compositions. Analytical polyacrylamide gel electrophoresis of the subunits at acid and alkaline pH values revealed heterogeneity. By free-zone electrophoresis at alkaline pH it was possible to isolate four discrete iso-forms of both the alpha and the beta subunit. All these eight individual subunits had activities consistent with those of their immediate precursor fractions. Isolation of electrophoretically homogeneous thyrotropin subunits has not been reported previously.


Proton magnetic resonance and conformational energy calculations of repeat peptides of tropoelastin. A permutation of the hexapeptide.

The conformation of a hexapeptide sequence occurring in tropoelastin is discussed from the results obtained using a combined approach of theoretical conformational energy calculations on HCO-Val-Ala-Prb-Gly-OMe and 1h nmr studies on t-Boc-Val-Ala-Pro-Gly-Val-Gly-OMe  in a dilute solution of methanol. Both studies have reasonable concurrence with respect to the preferred conformation of the hexapeptide and an analysis of the combined results suggests that the hexapeptide is stabilized by a beta-turn involving the Ala1,iC=O and Val4,iNH groups and a gamma-turn involving Gly5,iC=O and Gly3,iNH groups. A weaker interaction between Gly3,iC=O and Gly5,iNH groups is also found to be possible. Conformational features of the first valyl residue in the sequence Val-Ala-Pro-Gly-Val-Gly and the last valyl residue in Ala-Pro-Gly-Val-Gly-Val are compared and found to have similar torsion angles. The implications of such a similarity are discussed with respect to the conformation of the polyhexapeptide.


High affinity calcium binding peptides from the gills, gut and kidneys of the freshwater eel (Anguilla anguilla).

We report the isolation of high affinity calcium-binding peptides from homogenates of gills, gut and kidneys of the freshwater eel Anguilla anguilla. These peptides show identical gel-filtration profiles on Biogel P-6, from which a molecular weight of 3500 was estimated. The average binding constant (Kf) for calcium is 7 . 10(7)M-1, and MgCl2 up to 5 mM does not displace calcium.


Extraction of mitochondrial proteins by volatile anesthetics.

Heavy beef heart mitochondria were exposed to controlled concentrations of several volatile anesthetics including halothane, enflurane and chloroform. These anesthetics caused a concentration-dependent release of protein from mitochondria with maximal release occurring at 17.5% halothane and less release at lower and higher concentrations. The proteins released into the supernatants were analyzed by electrophoresis on slab gels containing a 6--20% gradient of acrylamide. The anesthetics caused the release of several polypeptides from mitochondria into the incubation medium; the major polypeptides released had molecular weights of 78 000; 48 000; 47 000; 43 000; 32 000 and 22 000. Two of these were identified by enzyme analysis and by co-electrophoresis with crystalline enzymes as the subunits of aspartate aminotransferase (43 000 daltons; EC 2.6.1.1) and malate dehydrogenase (32 000 daltons; EC 1.1.1.37). Mitochondria exposed to saturated halothane vapors were similar ultrastructurally to controls except that the halothane mitochondria appeared uncoupled. Similar results were obtained with O2 or N2 as carrier gas.


X-ray crystallographic and molecular orbital studies on the conformation of tryptamine.

The crystal structure of neutral tryptamine has been determined by X-ray methods. The refinements result in a conventional R value of 0.043. Tryptamine molecules are held together to form layered structures perpendicular to the c axis by van der Waals contacts and by N-H...N type hydrogen bonds. The conformation is similar to that of other cationic tryptamines. By the conformational energy calculation which was carried out by the Complete Neglect of Differential Overlap (CNDO/2) method, it is shown that the folded conformation observed in this crystal structure is attributed mainly to the nature of tryptamine molecule. Furthermore, it seems likely that this conformation is also significant and common in other numerous unsubstituted indolealkylamines, because their conformations are similar to that of the tryptamine.


The effect of ammonium chloride and glucagon on the metabolism of glutamine in isolated liver cells from starved rats.

1. Glucagon stimulated gluconeogenesis from glutamine in isolated liver cells to a far greater extent than that from any other amino-acid precursor. 2. Low concentrations of ammonium chloride (less than 1 mM) stimulated glucose production from glutamine. Glucagon further stimulated this glucose production, even in the presence of saturating concentrations of ammonium chloride. 3. In agreement with previous reports, glutamine hydrolysis by isolated mitochondria was found to be stimulated by ammonium chloride. It was found that ammonium chloride activated mitochondrial glutamine hydrolysis at the same concentrations at whict it stimulated glucose production from glutamine in liver cells. The effective activation of glutamine hydrolysis by ammonimum chloride in intact mitochondria was partially inhibited by rotenone and was abolished by uncoupling agents. 4. The addition of glucagon to hepatocytes metabolising glutamine led to a decrease in the intracellular concentration of glutamine and an increase in the intracellular concentration of glutamate. 5. It is likely that glucagon stimulates gluconeogenesis from glutamine by mechanisms which are additional to those that may operate in the stimulation of gluconeogenesis from other amino-acid precursors. It is suggested that both ammonium chloride and glucagon exert their effects on glutamine metabolism by increasing the effective activity of mitochondrial glutaminase (EC 3.5.1.2.).


The topology of epoxide hydratase and benzpyrene monooxygenase in the endoplasmic reticulum of rat liver.

The distributions of benzpyrene monooxygenase and epoxide hydratase in subfractions of liver microsomes from control and from phenobarbital- and methylcholanthrene-treated rats have been investigated. The specific activities of these enzymes in rough and smooth microsomes from control and phenobarbital-treated animals are approximately the same, whereas after methylcholanthrene treatment benzpyrene monooxygenase is four times higher and epoxide hydratase twice as high in the rough vesicles. Further subfractionation of rough and smooth microsomes by rate differential centrifugation revealed the distributions of both enzymes among microsomal vesicles to be highly heterogeneous. Comparison of these distributions leads to the conclusion that the benzpyrene monooxygenase system and epoxide hydratase may form a complex of unique stoichiometry in the membrane of microsomes from control rats, but that such a complex is not consistent with the distributions obtained after methylcholanthrene induction. Studies with proteases and the nonpenetrating chemical reagent diazobenzene sulfonate suggest that epoxide hydratase may be buried deeply in the hydrophobic phase of the membrane of the hepatic endoplasmic reticulum.


On the non-ferritin depot iron fraction in the rat liver.

Liver depot iron can be divided into two fractions: ferritin iron and non-ferritin depot iron. Three methods intended to measure the non-ferritin depot iron in the rat liver were compared using livers of normal rats and livers of rats loaded with iron by transfusion of erythrocytes. Liver depot iron varied between 75 and 850 microgram Fe/g liver. Non-ferritin depot iron, measured as the iron fraction sedimentable at 10 000 x g, was in the range 4--22 microgram Fe/g liver. This fraction did contain ferritin. When measured as the difference between total liver depot iron and heat-stable iron (ferritin iron), the range was 10--270 microgram Fe/g liver but this fraction also includes some ferritin iron. The values derived with both methods were linearly proportional to the total liver depot iron values. Non-ferritin depot iron, when measured as the difference between total liver depot iron and total ferritin iron, ranged from 0 to 190 microgram Fe/g liver. In this last method no ferritin iron is included. This method provides the best estimate of the non-ferritin depot iron fraction. The concentrations obtained with this method were not always linearly proportional to the total liver depot iron concentration. Intravenous injection of rat liver ferritin resulted in a rapid accumulation of ferritin iron in the liver, together with an increase of the non-ferritin depot iron fraction from 18 microgram Fe/g liver to 55 microgram Fe/g liver. This confirms a relationship between ferritin catabolism and the non-ferritin depot iron fraction.


Proton NMR relaxation study of the binding of pyridine and 1-methylimidazole to some ferriporphyrins and to metmyoglobin.

Proton spin-lattice relaxation times of pyridine and 1-methylimidazole complexed on Fe(III)protoporphyrin IX dimethyl ester, Fe(III)tetraphenylporphyrin in chloroform and on metmyoglobin in 2H2O have been measured. Caused by chemical exchange of the ligand molecules into the bulk solvent phase, the decay of their MZ-magnetization is given by a superposition of two exponentials from which the mean lifetime of the complexed species can be determined. This method offers the possibility to study exchange kinetics of ligand molecules from a well defined molecular configuration. The present data are compared with the results from linewidth measurements of the bulk phase molecules. From both sets of parameters a detailed picture of the ligand exchange can be gained, particularly if spin transition of the paramagnetic organic metal complex occurs, as is the case for some ferriporphyrins. For metmyoglobin, the NMR result is compared with relaxation times extracted from temperature jump experiments under similar conditions.


Sex difference in L-glutamine D-fructose-6-phosphate aminotransferase activity of mouse submandibular gland.

L-Glutamine D-fructose-6-phosphate aminotransferase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring), EC 5.3.1.19) activities in the three main salivary glands of male and female mice were measured. It was found that the activity in the submandibular gland was about 10 times more in females than in males, whereas the activities in the sublingual and parotid glands of males and females were similar. The activity in the submandibular gland of female mice was not affected appreciably by ovariectomy but it decreased to the level in males on injection of testosterone. The activity in males was not affected appreciably by injection of progesterone or 17beta-estradiol, but it increased to the level in females after castration. The increased activity in castrated male mice was decreased again to the normal level by testosterone injection. Thus, this sex difference is caused by androgen, not by female hormones. On the basis of in vivo experiments using actinomycin D, it was suggested that testosterone produced an "enzyme inhibitor", which suppressed the enzyme activity in the submandibular glands of androgen-rich animals.


Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids.

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticosteroids to the medium.  The degree of stimulation was directly related to glucocorticoid potency. Sex steroids, certain peptide hormones and prostaglandins E2 and F2alpha did not influence zinc accumulation. Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since 45Ca, 14C-labelled amino acids and [35S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatography confirmed that this additional cytosol zinc was bound to metallothionein. [35S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.


Effects of antihypertensive drugs on hepatic heme biosynthesis, and evaluation of ferrochelatase inhibitors to simplify testing of drugs for heme pathway induction.

Effects of a series of antihypertensive drugs on the activity of delta-aminolevulinate synthase and on the formation of porphyrins and cytochrome P-450 were examined in the 18-day-old chick embryo liver in ovo. Hydralazine, pargyline, phenoxybenzamine, clonidine, and spironolactone were found to induce delta-aminolevulinate synthase in this system. These drugs therefore have the potential to precipitate clinical expression in human hereditary hepatic porphyrias and should be avoided or used with caution in patients with these disorders. Differential effects of these and other drugs were observed in the avian liver, in that delta-aminolevulinate synthase was more commonly induced than were porphyrins and cytochrome P-450; the synthase was usually highest 6-12 h after injection, whereas porphyrins and cytochrome P-450 were highest at 24 h. Furthermore marked porphyrin accumulation was not seen with many drugs that induce delta-aminolevulinate synthase and cytochrome P-450 but was more characteristic of compounds that reduced the metabolism of protoporphyrin to heme, such as 1,4-dihydro-3,5-dicarbethoxycollidine (DDC) and high doese of hydralazine. A sensitive and convenient method to test for capacity to induce heme biosynthesis was adapted for use in the chick embryo liver. This employed a relatively small "priming" dose (0.25 mg) of DDC given with a drug being tested and a fluorometric assay of porphyrins in a liver homogenate obtained at 24 h. This simple method should facilitate screening for those drugs which induce the synthesis of delta-aminolevulinate synthase and/or cytochrome P-450 and are potentially dangerous to patients with hereditary hepatic porphyria.


Binding of N5,N10-methylene tetrahydrofolate and the inhibition of thymidylate synthesis by a folate-binding protein.

A mixture of two ionic forms of a folate-binding protein purified from chronic myelogenous leukemia cells reversibly binds N5,N10-methylene tetrahydrofolate and prevents the coupling of this cofactor to thymidylate synthetase in a terniary complex with fluorodeoxyuridylate. The binding protein also inhibits the enzymic synthesis of thymidine monophosphate by preventing the methylation of deoxyuridylate. These findings suggest that one function of the folate-binding protein may be to regulate the intracellular concentration of free folate cofactors and, thereby, modulate their functional activity.


Interactions between insulins and liver membrane receptors of guinea pig, calf and chicken. Exclusion of a species-specific insulin receptor.

Insulin binding experiments were performed with liver plasma membranes from guinea pig, calf and chicken. Bound insulin was separated from free insulin by a simple and rapid centrifugation of membranes through a layer of silicon oil. 125 I-labeled beef insulin was displaced from receptor sites by unlabelled guinea pig, beef and chicken insulin. The receptors of animals with insulins of different biological activity show similar basic characteristics and affinities to the different insulin molecules and thus are not specialised for the interactions with the homologous insulin molecule. The binding capacity of the membranes for beef insulin seems to be inversely related to the affinity of the homologous insulin to the receptor, guinea pig membranes showing the highest and chicken membranes the lowest receptor concentration.


Carbohydrate composition of cells and plasma membranes of Dictyostelium discoideum at selected stages of development.

Cells of Distyostelium discoideum representing four developmental stages were auto-analysed for constituent monosaccharides and their compositions compared. Rhamnose, ribose, fucose, glucose, mannose, galactose, glucosamine, galactosamine and an unidentified sugar were recovered after hydrolysis in 2 M HCl for 2 h at 100 degrees C. The relative proportions of the individual sugars were found to vary as a function of development. The largest variations were in the proportions contributed by galactose (from 2% of vegetative cell carbohydrate to 12% of the carbohydrate of fruiting bodies) and galactosamine (present in measurable quantity only in fruiting bodies). Plasma membrane "ghosts" were found to have the same monosaccharide constituents as whole cells, but in different proportions. Mannose contributed over 24% of the total carbohydrate recovered from aggregating cell "ghosts", but only 13% of carbohydrate recovered from "ghosts" prepared from vegetative cells. Galactose was the most abundant sugar recovered from vegetative "ghosts", and was second only to mannose in aggregating "ghosts".


Testosterone-induced proton release and inhibition of tryptic hydrolysis of albumin.

Further investigation of the effect of bound testosterone on the susceptibility of human serum albumin to tryptic hydrolysis has revealed a stimulation of hydrolysis at low binding levels, becoming an inhibition at higher binding levels. The inhibition, but not the stimulation, is associated with a two-stage steroid-induced proton release phenomenon which may involve the cysteine (SH) group and tyrosine residues. Proton release is also induced by Ca2+ and ethanol. The nature of the effect and the influence of Ca2+ suggests that stimulation of hydrolysis is associated with the presence of one molecule of testosterone and inhibition with the presence of 2-5 molecules at a set of sites which are thermodynamically equivalent.


Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen.

A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radioimmunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human and rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply.


A constant-volume ultrafiltration technique for the calculation of equilbrium binding data.

A constant-volume ultrafiltration technique is described, and details of its assessment presented. The retention characteristics of two membranes were evaluated using molecules of known molecular weight. The technique is rapid, precise, economical of material and yields equilibrium data. In these respects, it compares favourably with conventional techniques such as equilibrium dialysis.


[The hemoglobin molecule from a cybernetic and thermodynamic point of view (author's transl)].

The biological macromolecule hemoglobin (Hb) represents a reversibly functioning molecular machine. The Hb molecule is engaged in inner or structural work, which is cyclic in nature and is regulated through an exchange with the external milieu, of warmth and matter. The endothermic process of deoxygenation requires an external milieu that gives off warmth and that absorbs oxygen; the exothermic process of oxygenation, on the other hand, requires an external milieu that absorbs warmth and gives up oxygen. The Hb molecule exists in two extreme states: in a signal state for oxygenation and in a target state for deoxygenation. The transition from the signal to the target state occurs via the information process that is coupled to the structural work of the Hb molecule, whereby the absorbed warmth is converted via the structural work, without loss of energy, into the structural energy of the Hb molecule.


Visual detection of spatial contrast; influence of location in the visual field, target extent and illuminance level.

A model is proposed that permits the prediction of contrast detection thresholds for arbitrary spatial patterns. The influence of the inhomogeneous structure of the visual field and a form of spatial integration are incorporated in the model. A hypothetical density function for the spatial sampling units, which specifies the distribution of these units with respect to both size and location, is described. The density function is compared with anatomical and electrophysiological knowledge of the density of retinal and cortical receptive fields. This density function permits a particularly lucid interpretation in terms of pattern processing. It can be considered as a system that permits simultaneous global and focal views of the surroundings. Thedensity function, together with a schematized adaptation behaviour of single units, and an incoherent summation rule permit us to calculate a measure of the mass response, and consequently the threshold function. Predictions of the model are compared with recently obtained psychophysical data. In particular an explanation is offered for certain invariance properties of spatial contrast detection that seems to possess promising generality.


Physiological model analysis of involuntary human-voice tremor.

Frequency variations in the human voice result from voluntary and involuntary changes in the parameters of the vocal system. The present work deals with involuntary frequency perturbations from two theoretical aspects: 1) the influence of pitch period variations on frequency changes in the band-limited signal which results from the resonant characteristics of the vocal tract; 2) the physiological parameters of the vocal system which are potentially able to govern involuntary frequency changes. It is shown that the modulation function of the vocal-cord wave can theoretically be derived from its harmonics using FM demodulation techniques, and that higher distortion may appear at higher harmonics. It is also shown that involuntary geometirical changes of the vocal tract and its terminal impedance as well as tension and initial-area changes of the vocal cord--changes well within the physiological range--can influence frequency changes in the human voice. The present results are correlated with our reported experimental findings on involuntary voice tremor, used in psychological stress evaluation. The role of the central nervous system, and possible mechanisms for these phenomena, are discussed.


A model for light adaptation: producing Weber's law with bleaching-type kinetics.

An "adaptation model" having two stages is introduced and its mathematical properties are examined. The two stages are the "adaptive process" (parameter Kb), which has bleaching-type kinetics, and the "response function" (parameters Kr and n), which incorporates response saturation. In order to study the increment threshold functions generated by the "adaptation model" the concept of a "detector" is required. It is demonstrated that without an adaptive process the compression hypothesis, in the form of the "difference equation", produces increment threshold functions which saturate and do not obey Weber's law. It is then shown that an adaptive process with bleaching-type kinetics can prevent saturation and produce Weber's law behavior provided that the "adaptive strength" of the system exceeds the "detector sensitivity".


On the interaction between the central nervous system and the peripheral motor system.

The problem of the control of voluntary human movements is considered from a cybernetic point of view. The human motor system is considered to be divided into a central part and a peripheral part. The peripheral part is relatively well known and may be regarded as a set of subsystems with well known input-output relations. The interaction between the peripheral part and the central part is related to the mechanisms of the peripheral motor part. With regard to the central part two different types of control mechanisms are possible, a) an intricate functioning of the central part which generates the control signals with regard to internal and extrenal dynamical factors, b) the central part has some degree of independence with respect to the dynamics of the peripheral motor part. In the latter case the central part prescribes the desired movement exactly, but the final performance of the movement is also brought about by peripheral feed-back mechanisms. As a functional form of the interaction between the central part and the peripheral part it might be that the control signals are encoded in a way that is related to the muscle lengths.


Is the adaptation model a valid description of the vestibulo-ocular reflex?

The pendulum model of the vestibulo-ocular reflex, including the effects of adaptation, has been evaluated using the responses of 36 normal subjects to impulsive stimuli of 128 and 256 degree/s. Estimates of the model parameters such as the time constants, the slow velocity threshold, and the minimum stimulus required to produce an after-nystagmus have been obtained using a new analytical technique. Although some of the data support the validity of the adaptation model, evidence is presented to demonstrate that the overall applicability of the model is limited.


A study of the motor unit action potential by means of computer simulation.

In order to study the motor unit action potential a computer stimulation model was developed. It is based on the superposition of single muscle fibre potentials of the fibres belonging to the motor unit. The parameters which characterize each fibre (spatial position, diameter, and a dispersion of arrival time of the potential at the electrode) are chosen from statistical distributions which can be derived from anatomical and physiological data. The electrode type, position and dimensions can be specified. Simulated motor unit action potentials are presented in the time and frequency domain. The stimulation results refer to  (1) the influence of the electrode position and dimensions with respect to the motor unit territory, (2) the meaning of this model for the study of pathological phenomena, (3) the variability of some parameters characterizing the motor unit, (4) the selectivity of uni- and bipolar electrodes and finally (5) the influence of the geometrical situation of the motor end-plates within the muscle, on the shape of motor unit action potentials.


Anistropic connectivity and cooperative phenomena as a basis for orientation sensitivity in the visual cortex.

A computer simulation model of the neural circuity underlying orientation sensitivity in cortical neurons is examined. The model consists of a network of 3000 neurons divided into two functionally distinct cell types: excitatory (E-cells) and inhibitory (I-cells). We demonstrate that both orientation sensitivity and shape selectivity can be accounted for by making the following assumptions: 1) thalamic afferents to a sheet of cortical neurons are retinotopically organized; 2) thalamic afferents come from a single neuron, or at most a few neurons, in the lateral geniculate nucleus; 3) cortical activity is cooperative, i.e. largely dependent on intracortical connections, some of which have anisotropies along directions parallel to the pial surface. Anisotropies are specified only by the distribution of cells which are postsynaptic to a particular neuron, without specifying the axonal or dendritic contributions. In this paper, orientation sensitivity arises through cooperative interactions among neurons having anisotropic excitatory, and isotropic inhibitory connections.


Biological oscillators can be stopped--topological study of a phase response care.

Many biological oscillators are stable against noise and perturbation (e.g. circadian rhythms, biochemical oscillators, pacemaker neurons, bursting neurons and neural networks with periodic outputs). The experiment of phase shifts resulting from discrete perturbation of stable biological rhythms was developed by Perkel and coworkers (Perkel et al., 1964). By these methods, they could get important insights into the entrainment behaviors of biological rhythms. Phase response curves, which are  measured in these experiments, can be classified into two types. The one is the curve with one mapping degree (Type 1), and the other is that with zero mapping degree (Type 0) (Winfree, 1970). We define the phase response curve mathematically, and explain the difference between these two types by the homotopy theory. Moreover, we prove that, if a Type 0 curve is obtained at a certain magnitude of perturbation, there exists at least one lower magnitude for which the phase response curve cannot be measured. Some applications of these theoretical results are presented.


Psychopathy and electrodermal responses to nonsignal stimulation.

Electrodermal activity was monitored while 64 prison inmates were presented with a random series of fast and slow rise-time tones ranging in intensity from 80 to 120dB. The inmates were divided into groups on the basis of global ratings of psychopathy and scores on the Socialization (Os) scale. The group considered to be most psychopathic (high ratings of psychopathy and low So scores) gave smaller skin conductance responses than did the other groups, but only to the 120dB tones. These findings are consistent with the hypothesis that psychopaths are electrodermally hyporesponsive to intense nonsignal stimuli. High ratings of psychopathy were associated with slow recovery of the skin conductance response, but only with the 120dB fast rise-time tones, and only in the left hand.


Development of bidirectional operant modifications of the cephalic vasomotor response employing a positive reinforcer.

The purpose of the present investigation was to assess the effectiveness of a positive reinforcer in the bidirectional operant conditioning of the cephalic vasomotor response and to study the development, across sessions, of operant control over this response. To this end, 24 male, Caucasian subjects participated in five daily sessions (two shaping sessions and three operant control sessions) during which they were reinforced for producing either vasodilations (N = 12) or vasoconstrictions (N = 12) of the temporal artery. The results clearly demonstrated that the cephalic vascular responses can be brought under some degree of operant control employing a positive reinforcement paradigm and further that such control over cephalic vasoconstriction developed more rapidly than did operant control of cephalic vasodilation. Some evidence is presented to support a cognitive mediation effect.


Physiological response patterning in short--and long--term heart rate control training.

The present investigation assessed the physiological response patterns which accompany heart-rate control performance, both early and late in training. Six subjects were employed who received six heart-rate control training sessions, half of which were speeding sessions and half of which were slowing. Heart rate, skin resistance, frontalis EMG, systolic and diastolic blood pressure, and respiration rate were continuously recorded throughout all training sessions. Results indicated that the physiological response topography during heart-rate control performance changes from early to late training. Moreover, the changes are different for speeding and slowing performance, suggesting the involvement of different underlying physiological mechanisms in the two tasks.


Interhemispheric asymmetries in the visual evoked response: effects of stimulus lateralisation and task.

VER's were elicited by briefly presented lateralised letters in two experiments. In experiment 1 the subjects engaged in verbal processing of the letters and in experiment 2 they engaged in visuospatial processing. In both experiments the latencies of the first two components of the VER's were consistent with previous findings and with the anatomy of the retino-cortical visual pathways. The pattern of hemisphere asymmetries of the amplitudes of the middle and late components differed in the two experiments in that (i) in the second experiment the amplitude of the middle component from the left hemisphere did not vary with field of stimulus presentation although this was the case in the first experiment and (ii) a hemisphere asymmetry (left greater than right) was observed in the late component only in the second experiment. The results are interpreted as evidence of difference in the cerebral processing of the stimuli in respect of the task demands of the two experiments.


Circadian variation in performance, subjective sleepiness, sleep, and oral temperature during an altered sleep-wake schedule.

The effect of an altered sleep-wake schedule on the interrelation of oral temperature, performance, and sleepiness was studied in 38 male Naval volunteers who maintained a 60 min treatment--160 min testing schedule for 40 consecutive hrs. During the 60 min treatment portion of each epoch, 8 subjected napped, 10 subjects exercised, and 20 subjects rested in bed. Sleep measures (for the nap subjects), oral temperature, performance on several tests, and Stanford Sleepiness Scale ratings were obtained at 10 equidistant intervals throughout the 40-hr period. Within-subject correlations showed that minimum oral temperature was significantly associated with maximum nap sleep time, errors on a vigilance task, and sleepiness ratings. In the nap subjects, errors and sleepiness ratings were highest following naps with high total sleep time, suggesting that sleep was detrimental to immediately subsequent performance and alertness. The distribution and interrelation of temperature, errors, and sleepiness, however, was similar in the three groups; this indicated that the synchronous circadian variation in these measures was responsible for the apparent detrimental effect of sleep in the nap subjects. When the diurnal effect was removed by holding time of day constant, the correlations among the variables fell to near-zero, indicating no causal relationship among the variables independent of the circadian rhythm.


Magnetic relaxation analysis of dynamic processes in macromolecules in the pico- to microsecond range.

A formalism based on the theory of Markov processes and suitable for the analysis of multiple internal motions in macromolecules is presented. Computer calculations of specific motional models for (13)C nuclear magnetic resonance (NMR) relaxation, treated as special cases of the proposed formalism, demonstrate the potential of this approach for discriminating between different motional models on the basis of NMR relaxation data.


New electric field methods in chemical relaxation spectrometry.

New stationary relaxation methods for the investigation of ionic and dipolar equilibria are presented. The methods are based on the measurement of non-linearities in conductance and permittivity under high electric field conditions. The chemical contributions to the nonlinear effects are discussed in their static as well as their dynamic behavior. A sampling of experimental results shows the potential and range of possible applications of the new techniques. It is shown that these methods will become useful in the study of nonlinear responses to perturbation, in view of the general applicability of the experimental principles involved.


Response of acetylcholine receptors to photoisomerizations of bound agonist molecules.

In these experiments, agonist-induced conductance is measured while a sudden perturbation is produced at the agonist-receptor binding site. A voltage-clamped Electrophorus electroplaque is exposed to trans-Bis-Q, a potent agonist. Some channels are open; these receptors have bound agonist molecules. A light flash isomerizes 3(-35)% of the trans-Bis-Q molecules to their cis form, a far poorer agonist. This causes a rapid decrease of membrane conductance (phase 1), followed by a slower increase (phase 2). Phase 1 has the amplitude and wavelength dependence expected if the channel closes within 100 mus after a single bound trans-Bis-Q is isomerized, and if the photochemistry of bound Bis-Q resembles that in solution. Therefore, the receptor channel responds rapidly, and with a hundred-fold greater closing rate, after this change in the structure of a bound ligand. Phase 2 (the conductance increase) seems to represent the relaxation back toward equilibrium after phase 1, because (a) phase 2 has the same time constant (1(-5) ms) as a voltage- or concentration-jump relaxation under identical conditions; and (b) phase 2 is smaller if the flash has led to a net decrease in (trans-Bis-Q). Still slower signals follow: phase 3, a decrease of conductance (time constant 5(-10 ms); and phase 4, an equal and opposite increase (several seconds). Phase 3 is abolished by curare and does not depend on the history of the membrane voltage. We consider several mechanisms for phases 3 and 4.


Hapten-linked conformational equilibria in immunolglobulins XRPC-24 and J-539 observed by chemical relaxation.

The interaction of oligogalactan haptens with the murine myeloma proteins XRPC-24 and J-539 has been investigated by the fluorescence temperature-jump method. The relaxation spectrum is composed of two processes, the faster representing hapten assocaition and the slower a protein isomerization. In both cases the concentration dependence of relaxation times and amplitudes was consistent with the general mechanism formulated by Lancet and Pecht (1976, Proc. Natl. Acad. Sci. U.S.A. 73:3549), in which the equilibrium between two conformations of the protein is shifted by hapten binding. The intact proteins and their Fab fragment had identical kinetic behavior, indicating that the conformational changes are located in the Fab region. Temperature dependence analysis for protein J-539 permitted the calculation of activation parameters and led to a consistent energy profile for all the elementary steps. The conformational states are separated by large activation barriers, but have similar free energies. The results suggest that hapten-induced conformational changes in immunoglobulins are more general phenomena than was previously thought.


The rate of entry of dioxygen and carbon monoxide into myoglobin.

The model for carbon monoxide or dioxygen recombination with heme proteins developed by the group at the University of Illinois is reexamined. We propose that the carbon monoxide or dioxygen molecule enters the protein at essentially a diffusion-limited rate determined by the solvent viscosity and that the protein offers no important barriers to this entry. The viscosity dependence of the entry rate k(ED), its magnitude (1 x 10(10) M(-1)s(-1), and the rate of quenching of triplet states of protoprophyrin IX in apomyoglobin by dioxygen are used as supporting evidence. Comparison is made to the model of a fluctuating protein developed by G. Weber.


Single cell observations of gas reactions and shape changes in normal and sickling erythrocytes.

Microspectrophotometry has been applied to single red blood cells to reinvestigate the linked processes of diffusion of gases inside the erythrocyte and their combination with hemoglobin. The experiments took advantage of the photosensitivity of the cabron monoxide derivative of hemoglobin, which allows ligand release from the CO-saturated red cells under strong illumination and recombination when the light is switched off. The photochemical method was also used to study the kinetics of sickling on ligand removal in single erythrocytes of Hb S carriers. The results give new information on the mechanism of the sickling process.


Detection of hindered rotations of 1,6-diphenyl-1,3,5-hexatriene in lipid bilayers by differential polarized phase fluorometry.

Differential polarized phase fluorometry has been used to investigate the depolarizing motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the isotropic solvent propylene glycol and in lipid bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC), dipalmitoyl-L-alpha-phosphatidylcholine (DPPC), and other phosphatidylcholines. Differential phase fluorometry is the measurement of differences in the phase angles between the parallel and perpendicular components of the fluorescence emission of a sample excited with sinusoidally modulated light. The maximum value of the tangent of the phase angle (tan Delta(max)) is known to be a function of the isotropy of the depolarizing motions. For DPH in propylene glycol the maximum tangent is observed at 18 degrees C, and this tangent value corresponds precisely with the value expected for an isotropic rotator. Additionally, the rotational rates determined by steady-state polarization measurements are in precise agreement with the differential phase measurements. These results indicate that differential phase fluorometry provides a reliable measure of the probe's rotational rate under conditions where these rotations are isotropic and unhindered.Rotational rates of DPH obtained from steady-state polarization and differential phase measurements do not agree when this probe is placed in lipid bilayers. The temperature profile of the tan Delta measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Delta at the transition temperature (T(c)), followed by a gradual decline in tan Delta at temperatures above T(c). The observed tanDelta(max) values are only 62 and 43% of the theoretical maximum. This defect in tanDelta(max) is too large to be explained by any degree of rotational anisotropy. However, these defects are explicable by a new theory that describes the tan Delta values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r(infinity). Theoretical calculations using this new theory indicate that the temperature dependence of the depolarizing motions of DPH in these saturated bilayers could be explained by a rapid increase in its rotational rate (R) at the transition temperature, coupled with a simultaneous decrease in r(infinity) at this same temperature. The sensitivity of the tan Delta values to both R and r(infinity) indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.


The structure of the retinylidene chromophore in bathorhodopsin.

Resonance Raman data on bathorhodopsin (bovine and squid) at 95,77, and 4 degrees K support a mechanism of excitation proposed by Lewis in which both a protein conformational transition and chromophore structural alteration to a "dicisoid" configuration are required to generate the bathorhodopsin species observed in steady-state photostationary mixtures. However, these results also suggest that the molecular structure with a red-shifted chromophore absorption detected at room temperatures in 1 ps using picosecond absorption spectroscopy may not necessarily have the same chromophore conformation as the steady-state bathorhodopsin species.


Probing ultrafast biological processes by picosecond spectroscopy.

A brief discussion of the initial events leading to the visual transduction process will be presented to illustrate the capabilities of picosecond spectroscopy.


Application of pulse radiolysis to the study of proteins: chymotrypsin and trypsin.

The one-electron reduction of chymotrypsin, trypsin, and their zymogens have been studied by pulse radiolysis. The optical spectra of the transient products from the two active enzymes display a pH-dependent band at 360 nm, associated with the histidine-electron adduct. The yield of the histidyl radical as a function of pH is consistent with a pK(a) less than 4.5, which suggests that the radical is located at the enzyme active site. The histidines of the proenzymes chymotrypsinogen and trypsinogen are unreactive towards the hydrated electron. We conclude that formation of the histidine-electron adduct at the serine protease active site is sensitive to the physical alterations which accompany protease activation.


Photo-initiated ion formation from octaethyl-porphyrin and its zinc chelate as a model for electron transfer in reaction centers.

Ion formation from the reaction of triplet (T) and ground state (P) octaethyl-porphyrin (OEP) and zinc octaethyl porphyrin (ZnOEP) and the corresponding cross-reactions have been measured in dry acetonitrile. A uniquely sensitive and fast conductance apparatus and a pulsed dye laser allowed the measurements to be made at the necessarily very low concentrations of T. The hemogeneous reaction of T (ZnOEP) and P (ZnOEP) occurs with rat constant k(1) = 2.0 x 10(8) M(-1)s(-1) and an ion yield of 67%. The similar homogeneous reaction of OEP has k(2) = 1.3 x 10(8)M(-1)s(-1) but an ion yield of only 3%. The cross-reaction of T (OEP) with P (ZnOEP) has k(3) = 1.5 x 10(8) M(-1)s(-1) and an ion yield of 27%, while the inverse cross-reaction of T (ZnOEP) with P (OEP) has k(4) = 3 x 10(8) M(-1)s(-1) and an ion yield of 20%. Thus, the rate constants are only slightly affected but the yields are sensitive to the porphyrin. The possible formation of the heterogeneous ions ZnOEP+ + OEP-, thermodynamically favored by 0.3 V over the homogeneous ions, has little influence on the observed yields. The data are explained by electron transfer and Coulomb field-electon spin-controlled escape of the initial ion-pair.


A picosecond pulse train study of exciton dynamics in photosynthetic membranes.

The fluorescence decay time of spinach chloroplasts at 77 degrees K was determined at 735 nm (corresponding to the photosystem I emission) using a train of 10-ps laser pulses spaced 10 ns apart. The fluorescence lifetime is constant at congruent to 1.5 ns for up to the fourth pulse, but then decreases with increasing pulse number within the pulse train. This quenching is attributed to triplet excited states, and it is concluded that triplet excitons exhibit a time lag of about 50 ns in diffusing from light harvesting antenna pigments to photosystem I pigments. The diffusion coefficient of triplet excitons is a least 300--400 times slower than the diffusion coefficient of singlet excitons in chloroplast membranes.


An inexpensive microcomputer-based stopped-flow data acquisition system.

A low-cost (less than $2,500) microcomputer-controlled data acquisition system for use with a stopped-flow instrument  is described. Data acquisition, reduction, signal averaging, kinetic modeling, and plotting are performed under software control. Applications to biological and inorganic systems are presented.


Picosecond fluorometry in primary events of photosynthesis.

Many laboratories in different countries are involved in the study of the mechanism of conversion of light energy into chemical energy, namely photosynthesis. As is evident from the literature, the initial phases of photosynthesis, which determine the character of this process, proceed at time intervals of 10(-8) and 10(-13) s. They are associated with absorption of light quanta and energy transfer from the molecules of light-harvesting antenna (LHA) chlorophyll and accesory pigments to the reaction centers (RC), where the key reaction of photosynthesis occurs: photo-induced charge separation. Evidently it is of importance to study experimentally the process that occurs within the 10(-8) -10(-13)s time domain.


1-N-Acetylspermidine: occurrence in normal human serum.

Using gas chromatography mass spectrometry following derivatization (N-trifluoroacetylation), 1-N-acetylspermidine was found to occur in normal human serum at a level of approximately 0.008--0.5 nmol ml(-1), i.e. about two orders of magnitude lower than the corresponding spermidine level. No evidence for the occurrence in serum of the isomeric 8-N-acetylspermidine was found. Mass spectra of authentic samples of trifluoroactylated 1-N- and 8-N-acetylspermidine are presented.


Field desorption mass spectrometric analysis of organic compound residues in the environment. I--Organochlorine insecticides.

Field desorption mass spectrometry is applied to the positive identification of organochlorine insecticides and their related compounds residing in field soil environment. Additionally, standard field desorption mass spectra of these compounds are presented. Soil samples were collected in lettuce and spinach fields, and insecticides were extracted, cleaned up, and separated with thin-layer chromatography. Residue levels were measured by injecting the extract into a gas chromatography equipped with an electron capture detector. Residues of p,p'-DDT and dieldrin in field soil samples were clearly identified by field desorption mass spectrometry using the emitter dipping method. Moreover, mixed residues of these insecticides were simultaneously confirmed. However, residues of insecticides and their related compounds other than p,p'-DDT and dieldrin could not be characterized. All spectra of standard showed prominent [M].+, [M(35Cl(n-1), 37Cl)].+ and [M(35Cl(n-2, 37Cl2)].+ ions, and the [M(35Cl(n-1), 37Cl)].+ ion was the base peak in all spectra obtained.


Electron impact and chemical ionization mass spectrometry of steroidal spirolactones.

Electron impact and chemical ionization mass spectra are reported for several steroidal spirolactones and their TMS ethers. The electron impact spectra were characterized generally by low abundance molecular ions and large numbers of fragment ions. Methane chemical ionization spectra exhibited high intensity [M+H]+ and/or [M+H-H2O]+ or [M+H-TMSOH]+ ions with few other fragment ions. Ammonia chemical ionization spectra had intense [M+H]+ and/or [M+NH4]+ ions with a few fragment ions generally formed by loss of H2O  or TMSOH from these parent ions. Ammonia chemical ionization gave intense parent ions even for polyhydroxy compounds and their TMS ethers in contrast to methane chemical ionization. The results of this study suggest that a combination of electron impact with ammonia chemical ionization mass spectrometry would offer the best techniques for detection and identification of these compounds in biological fluids.


New derivatives of prostaglandin A1 and specific detection of prostaglandin A's and 190hydroxylated prostaglandin A's in human semen.

The derivatization of prostaglandins of the A series with 1:1 mixtures of bis-(trimethylsilyl)trifluoroacetamide and nitrogen-containing non-aromatic heterocyclics such as piperidine, pyrrolidine, morpholine and hexamethylenimine (1--4 h at 60--70 degrees C) gives new types of derivatives, designated as 11-heterocycle, 9-enol PGA (TMS)3. These derivatives show very simplified and characteristic mass spectral patterns strikingly dominated by a common [M-173]+ fragment ion and easily detectable by selected ion monitoring. This feature allows the concurrent analytical detection of both prostaglandin A's and 19-hydroxy prostaglandin A's in biological samples. In this case 2 ml samples of human semen were extracted by direct ultrafiltration on a Pellicon membrane with a nominal molecular weight limit of 1000. The prostaglandins in the approximately or equal to 1.6 ml of ultrafiltrate thus obtained were recovered in ethyl acetate, derivatized as indicated above and detected by monitoring of the corresponding [M-173]+ ions.


Determination of lithium in microlitre amounts of human body fluids at therapeutic and normal levels by stable isotope dilution and field desorption mass spectrometry.

The use of field desorption mass spectrometry for the determination of lithium in body fluids at therapeutic levels--ppm region--as well as at the normal level--ppb region--has been developed. The use of a stable isotope enriched internal standard, together with the outstanding sensitivity of field desorption for alkali cations and the high specificity of mass spectrometry, allows a quantitative determination of lithium in microlitre amounts of body fluids, such as plasma, saliva and urine. The assay allows a determination of lithium even at ultratrace concentrations where routine spectroscopic procedures cannot be applied. Analysis of plasma required a simple protein precipitation, whereas saliva and urine could be analysed without treatment. The precision of the data obtained ranged from 2--10%. The time consumption for one analysis in routine work is about 20--30 min.


A selected ion monitoring method for the determination of pethidine and norpethidine in plasma. Comparison with a gas chromatographic method using electron capture detection.

An analytical procedure for the simultaneous determination of pethidine and norpethidine in plasma by selected ion monitoring is described. The method has a capacity of 15--20 analyses per hour and can be used to determine pethidine and norpethidine down to 25 ng ml(-1) and 5 ng ml(-1), respectively. The selected ion monitoring method has been compared with a method based on electron capture detection after analysis of pethidine and norpethidine in spiked plasma samples and in plasma from patients. The two methods are capable of performing selective and accurate determinations of pethidine and norpethidine, in the concentration ranges obtained in man after a single therapeutic dose of pethidine.


Mass spectral studies on prostaglandins. V--Deuterated analogues of prostaglandin F2alpha.

In order to check the fragmentation mechanisms proposed in the preceding paper of this series the mass spectral behaviour of some deuterated analogues of prostaglandin F2alpha have been studied. In general, the investigation of these compounds confirmed the previous assumptions and shed light on some further details of the fragmentation pathways.


Metabolic N-oxidation of 3-substituted pyridines: identification of products by mass spectrometry.

The mass spectral characteristics of the N-oxides of a range of 3-substituted pyridines, and of quinoline and isoquinoline, are described. The molecular ion is the base peak in the majority of cases, provided that thermolysis is minimized when using the direct probe or gas chromatography inlets. Chromatographic and mass spectral evidence is presented which indicates that biological oxidation of the heteroaromatic nitrogen of 3-substituted pyridines is a route of metabolism in vivo and in vitro.


Selected metastable peak monitoring: a new specific technique in quantitative gas chromatography mass spectrometry.

Using the combined electrostatic analyser-magnet scan facility of a double focusing mass spectrometer, the daughter ion peak for the fragmentation, [M-57]+ leads to [M-57-76]+, of a 5alpha-dihydrotestosterone tert-butyl-dimethylsilyl ether was monitored during gas chromatography mass spectrometry. The detection limit was approximately 20 pg. The steroid was detected by this method in an extract of human blood plasma.


Dissimilatory nitrate reduction by liquid membrane encapsulated cell-free extracts and whole cells of Micrococcus denitrificans.

The combination of Sephadex G-15 and ion-exchange resin columns allows one-step desalting and separation of cellodextrins using water as the sole eluent. The column apparatus described in this paper has the potential of producing up to 3 g cellodextrins in one day. In addition, the columns described are stable and do not require repacking or regeneration after each run. Hence the potential exists for scaling up this system for even greater production of cellodextrins if need be.


[Effect of feeding on the activity of duodenal smooth muscle in dogs].

The electric activity of the duodenum smooth muscles was investigated with chronically implanted electrodes. This activity after feeding was compared with that in fasting during the time equal to the period of rest and the active period of the duodenum in fasting. The number of pacesetter potentials was identical during digestion and fasting. The number of spike potentials was significantly different during these compared states. The relationship of the "digestive" and "hungry" electrical activities of the duodenum depended both on the compared type of potential and on the compared time periods. Apparently the optimum of the "digestive" function of the duodenum smooth muscles consists in the fact that this activity fluctuates within a definite range--between the maximum level (during the active period) and the minimum one (during the period of rest, i.e. fasting).


[Activity in the myelinated fibers of a cat cutaneous nerve in response to cooling].

The afferent activation in cat cutaneous nerve fibres in response to cooling was analyzed using the collision method and the method improving the signal-to-noise ratio in the antidromic action potential neurogram. It was found that the nerve fibres of groups Adelta1 and Adelta2 were activated under cutaneous receptors cooling. The activity of a small group of nerve fibres with conduction velocity 13.0--7.5 m/s was inhibited in response to cooling. A group of "mixed" fibres inhibited mainly its activity, but only an insignificant part of these fibres activated in response to cutaneous receptors cooling.


[Alteration of the antigenic properties of the synaptic membrane proteins of rat cerebral cortex during learning].

Rabbit antisera to the synaptic membrane fraction (SMF) of the cerebral hemispheres of control rats and animals with conditioned avoidance behaviour, reacting with the corresponding antigens in the complement fixation test, were obtained. Antibodies reacting specifically with the SMF of the brain of trained animals were revealed in the "trained" serum after the removal of antibodies to heterologous antigens and to the control SMF antigens.


[Heat release and body temperature of white rats in the presence of increased oxygen tension].

Experiments were conducted on bodies warmed under constant regimen imitating animals; the effect of hyperoxybaria at 1.2--4.0 atmospheres on the heat flow (HF) from them and their temperature was studied. An increase of the recorded HF with its subsequent progressive decrease during the continuing exposure to high gas pressure was revealed. At the same time a fall of the bodies' temperature was observed; this indicated an increased heat loss. The periods of gas compression and decompression were accompanied by a marked increase and decrease of the HF, respectively. In experiments on albino rats alterations in the HF level under compression and decompression were less, and the HF level during the hyperbaria remained almost constant; the rectal and subcutaneous temperature showed no statistically significant difference from the original one. It is suggested that this may be the result of the thermogenesis rise.


[Glycogen metabolism in ischemic organs].

Acute ischemia was created by placing a tourniquet on the extremity or on a clamp on the kidney limb for a period corresponding to the critical metabolism level in the test tissue study. Restoration of circulation in the ischemic kidney led to the excessive accumulation of glucose high-molecular polymer of the glycogen type. The character of its branching in the molecule determined by the iodine complex spectrum pointed to the changes in the processes of glycogen biosynthesis. Lactate of the ischemic kidney could be used for the glycogenesis requirements. This anomalous glycogen was shown to be actively uptaken by the kidney tissue. Glycogen accumulation in the muscle tissue following acute ischemia failed to exceed the normal level, and its structure was unchanged.


[Experimental analysis of the humoral mechanism of microcirculatory disorders following thermal and mechanical traumatic shock in experiments on parabiotic rats].

In experiments on parabiotic rats with crossed circulation and separate innervation mechanical or burn trauma of one of the partners was followed by shock-like condition of the other one, and by microcirculatory changes in the mesentery typical of burn or traumatic shock. It shows that in burn shock, as well as in traumatic one, the detected changes in microcirculation are due to humoral, but not to neural factors.


[Effect of cerebral ischemia on the metabolism of individual lipid fractions].

Ninety minutes after ligation of common carotid arteries a decrease of the metabolism intensity both of the total phospholipids (PL) and of their different fractions was nearly the same. In the diencephalon reduction of the metabolic rate of the total PL was accounted for by metabolic depression of phosphatidylcholine and phosphoinositide fractions only.


[Effect of artificial gravitational force in space flight on the concentration of water soluble proteins in nerve tissue structures].

The content of water-soluble proteins in the grey and white matter of the spinal cord, spinal ganglia, and the sensory-motor area of the large hemispheres of the brain of rats after a 18.5-day space flight was measured. The content of water-soluble proteins in the grey and white matter of the spinal cord and spinal ganglia of rats exposed to weightlessness showed a significant decrease 4.5--9.5 hours postflight. The water-soluble protein content in the white matter of the spinal cord diminished in rats exposed to artificial gravity inflight. The water-soluble proteins content in the grey matter of the spinal cord of rats exposed to weightlessness increased significantly 25 days postflight. The content of water-soluble proteins in the grey matter of the cortex of the sensory-motor area of the large hemispheres of the brain of rats exposed to weightlessness and artificial gravity remained unchanged in comparison with the vivarium controls both 4.5--9.5 hours and 25 days postflight.


[Structuro-functional changes in dog liver and regional lymph node lysosomes in toxic hepatitis].

Structural and functional changes in the dog liver and regional lymph nodes lysosomes were studied during toxic hepatitis induced by CCl4 administration (single and repeated). Total activity of lysosomal enzymes (acid RNA-ase and beta-galactosidase) was higher in the regional lymph nodes than in the liver, reflecting the barrier, protective function of the organ. During acute toxic hepatitis the specific activities of acid RNA-ase and cathepsin D displayed a sharp rise. No normalization of the indices under study occurred during the observation period (from 8 to 30 days). At the same time there was a rise of the regional lymph node weight and an elevation of the relative macrophage and neutrophil content in the sinuses. The increased activity of the lysosome enzymes in the regional lymph nodes in injury of the liver was connected with greater functional load on the lymph nodes effecting hydrolysis of biopolymeres which penetrated into the regional lymphatic node with the lymph.


[Effect of cocaine molecule fragments on the central nervous system].

The influence of ecgonine, tropine, tropinon, and some of their derivatives, propan, N-methylpyrrholidine, N-methylpiperidine on impulse summation in the central nervous system, conditioned reflex of avoidance, antagonism to hexenal, synergisim to cocaine and also their toxicity (LD50) have been studied under experimental conditions. As shown, benzoyl-ecgonine, ecgonine methyl ester, ecgonine, tropine, pseudotropine, carbomethoxytropinon, tropinon, tropine, tropan N-methylpyrrolidine, and N-methylpiperidine produced a pronounced stimulating effect on the central nervous system activity; in this respect they were similar to cocaine. The following conclusions were made: the substances whose molecules contained tropan structure or one of its fragments possess the central stimulating effect. The stimulating effect of cocaine on the central nervous system could be due to the tropan fragment of its molecule.


[Effect of methylapogalanthamine on the tone of cerebral, extracranial and peripheral vessels].

It was established in acute experiments on anesthetised and nonanesthetised cats by means of resistography that methylapogalanthamine decreased the total arterial pressure, the tone of cerebral vessels, extracranial and femoral vessels. The effect of the latter was more pronounced. A two-phase reaction (dilatation-constriction) was not rare. Methylapogalanthamine diminished the constriction reaction of intra- and extracranial vessels in response to st mulation of the cervical sympathetic nerves. The vasodilator action of methylapogalanthamine was enhanced by anesthesia and decreased by rausedyl.


[Effect of reversible inhibitors on the reaction capacity of functional portions of the active center of acetylcholinesterase].

The effect of galanthamine, tacrine, and oxazyl (ambenomum) on human red cells acetylcholinesterase phosphorylation by armine and Gd-42 (o-ethyl-s-beta-ethylthioethyl ester of methylthiophosphinic acid) was studied. In the presence of galanthamine phosphororganic inhibitors interacted only with the active center of the enzyme, the anionic site of which was not occupied by the reversible inhibitor. Tacrine and oxazyl decreased the reactivity of the free enzyme and the rate of its phosphorylation.


[Kinetics of guinea pig taenia coli smooth muscle relaxation. Effect of papaverine and polarization].

Experiments were conducted on isolated strips of the guinea pig taenia coli by the double sucrose gap technique. The effect of papaverins on the relaxation kinetics of the polarized and depolarized smooth muscle after contractions induced by electric stimulation was investigated. Depolarized muscle was stimulated by long-lasting hyperpolarizing pulses; contraction arose in response to the termination of the current pulse (off-response). It was found that: 1) the relaxation phase of the depolarized muscle off-response was slowed down as compared with the relaxation phase of the spontaneous contractions in Krebs solution; 2) papeverine accelerates the relaxation phase of the off-response of the depolarized muscle; 3) papaverine does not accelerate the relaxation phase of the evoked contractions in Krebs solution; 4) hyperpolarization and additional depolarization accelerate the relaxation phase of the off-response of the depolarized muscle; 5) hyperpolarization does not accelerate the relaxation phase of the off-response of the depolarized papaverine-treated muscle. The data obtained suggest the existence of the slow electroexcitable calcium channels in the membrane of the smooth muscle cells blocked by papaverine. The calcium influx through these channels slows down the relaxation phase of the depolarized muscle off-response.


[Comparative findings on the stability of plasmid resistance to antibiotics in vitro and in vivo].

The authors carried out comparative study of the incidence of spontaneous plasmide-negative variants occurrence in the S. aureus 8325phi IIde population developing under various conditions in vitro and in vivo. The mean percentage of such variants in the population developing in vitro constituted 0.9 +/- 0.2 in broth, 1.2 +/- 0.3 in the exudate, 6.3 +/- 2.0 in the population developing in vivo in the subcutaneous purulent-inflammatory focus, 9.5 +/- 2.6 in the kidneys. The mean percentage of plasma-negative variants in the purulent exudate in vivo was significantly greater than in the purulent exudate in vitro. The incidence of negative variants occurrence in vivo depended on the macroorganism properties.


[Increase in the content of thymus hormone sensitive rosette-forming cell precursors in the spleen of mice vaccinated against smallpox].

Smallpox vaccine injection to 2-month-old C57BL/6j female mice caused during the first days significant rise in the number of precursor cells of rosette-forming lymphocytes, sensitive to the differentiating thymus extract effect. The number of these cells returned to the normal level by the 10th day after vaccination.


[Carcinogenicity of orthoaminoazotoluene for mouse intestines].

Carcinogenic activity of orthoaminoazotoluene in relation to intestinal tissues of A/He mice was revealed. Intestinal tumours developed in 19 of 60 mice given this carcinogen; the tumours were localized in the cecum and represented adenomas and adenocarcinomas secreting mucus.


[Characteristics of the mitotic cycle of cells during the terminal stage of development of ascitic hepatoma 22A].

The authors studied growth peculiarities of the ascitic hepatoma 22A at the terminal stage of development. As shown by the autoradiography method, some of the cells could be at the G1-period or at the R1-period for 2 or even 4 days; the average duration of the G2-period constituted 16 hours. 55--60% of the cells were at the much prolonged G1- and R1-period, 7%--at the S-period, and 9%--at the G2-period. The rest 25--30% of cells apparently leave the mitotic cycle irreversibly since they take no part in proliferation after the division stimulation.


[Effect of physical training on restoration of rat liver following partial hepatectomy].

Swimming training of rats led to a change of functional condition of hepatocytes accompanied by the changes in nuclei distribution by classes of DNA ploidity. The blood sugar level and glycogen content in the liver of trained rats were much greater than in the untrained animals 20 and 48 hours after partial hepatectomy. Reparative regeneration following partial hepatectomy in trained rats was characterized by a peculiar distribution of nuclei by classes of DNA ploidity and by entrance into the synthesis of diploid nuclei only, 20 hours after the operation.


[Effect of extracts of different origin on primary cultures of cells from inbred mice].

The effect of allogeneic and syngeneic extracts from inbred male and female mouse spleens on the primary culture of fibroblasts obtained from subcutaneous connective tissue of CBA and C57BL/6j female mouse fetus was studied. Cytotoxic and growth-inhibiting effect on the cultures increased successively in using extracts from the tissues of syngeneic male mice, allogeneic female and male mice. Consequently, an increase in the degree of antigenic differences of the target=cells and extracts led to enhancement of the allogeneic inhibition phenomenon. For the first time it was shown that in the syngeneic system the extracts from male mouse tissues (containing weak H-Y antigen) exerted a cytotoxic action on the inbred female mouse cells, i.e. caused reaction similar to allogeneic inhibition.


[Autoradiographic study of stimulation of RNA synthesis in fibroblasts].

The rate of uridine-5 3H incorporation into the skin-muscle wound fibroblasts of control and experimental animals, and also into the fibroblasts of flocculent connective tissue of intact animals was studied. The character of incorporation and distribution of the isotope in the nuclei and cytoplasm in the flocculent connective tissue fibroblasts differed in intact mice from those in the wound fibroblasts only by intensity. Potassium orotate RNA synthesis stimulation in the fibroblast nuclei closely correlated with the increased rate and intensity of the labeled RNA passage from the cell nuclei to the sites of collagen synthesis.


[Migration of hematopoietic stem cells in thermal burns].

Experiments on mice (CBA X C57BL) F1 have shown that a high level of endogenous glucocorticoids in the blood 30 min to 6 hours after burn is accompanied by a 2--2 1/2-fold decrease of the circulating CFU. At this period CFU migration from the bone marrow area shielded during the irradiation (850 R) is inhibited. The stem cells migration is increased 3--4 days after the burn.


[Organ cultures of embryonal rat brain tissue (hippocampal area) in a study of the transplacental effect of nitrosoethylurea].

The transplacental action of nitrosoethylurea (NEU) was studied in organic cultures of the embryonic rat brain tissue (the hyppocampus region). The brain tissue proved to be highly sensitive to NEU. This was expressed in a greater survival of the experimental cultures in comparison with the control and in the appearance of epithelial proliferations of the vascular plexus resembling, in individual cases, adenomas. The amount of morphological changes observed in the experiment depended on the carcinogen dose.


[Effect of several extreme influences on the submicroscopic organization of rat liver following carbon tetrachloride poisoning].

Submicroscopic structure of parenchymatous cells was investigated in the liver of rats given CCL4 (0.12 ml/100 g body wt) for 25 days. After cessation of this treatment the animals were subjected to the action of extreme stimulants (immobilization or overheating under sunlight or both). CCL4 poisoning led to the appearance of many lipid incorporations in the cytoplasm of hepatocytes serving as the basis for fat infiltration in the liver. The action of extreme factors was followed by accumulation of lipids in the lumina of the granular cytoplasmic reticulum never found in any other pathological conditions, and apparently reflecting profound disturbances of intracellular metabolism.


[Cytophotometry of DNA in isolated mouse heart myocytes].