The structural organization of nuclear messenger RNA precursor. I. Reassociation and hybridization properties of double-stranded hairpin-like loops in messenger RNA precursor.

The hybridization and renaturation properties of double-stranded hairpin-like loops isolated from giant nuclear messenger RNA precursor of mouse liver or ascites carcinoma cells were studied. About half of the hairpins in messenger RNA precursor appear to contain similar sequences, as indicated by the very fast kinetics of renaturation of the denatured double-stranded RNA sequences. These sequences have no tissue specificity. About one third of the hairpin sequences can hybridize to messenger RNA. It is suggested that the long hairpins in messenger RNA precursor play the role of sequences separating messenger RNA sequences from non-informative sequences and that these hairpins are recognized by processing enzymes.


Dielectric increment of low molecular weight RNA in dioxane after application of static electric fields.

A soluble RNA with a molecular weight between 15 000 and 23 000 was extracted from calf thymus and chromatographically purified. The RNA was slightly soluble in doxane (approx. 2.5 mug/ml) and RNA-dioxane solutions were used to fill a cell (452.02 pF capacitance) to which static electric fields of variable strength were applied. The dielectric permittivity of the solutions was measured at a fixed time interval after the application of the electric field. The RNA solutions showed a dielectric increment proportional to the strength of the applied field and to the RNA concentration. The existence of a hystersis effect was proven and the effect of the electric field on the RNA molecule might be related to a dielectric saturation phenomenon parallel to long-term changes of the molecule.


Permanganate ion oxidations. IX. Manganese intermediates (complexes) in the oxidation of 2,4(1H,3H)-pyrimidinediones.

Uniquely stable manganese intermediates (complexes) are formed from the permanganate ion oxidation of the 5,6-carbon-carbon double bond in several 2,4(1H,3H)-pyrimidinediones [uracil, (compound 7), 5-methyluracil (thymine, compound 5), and 6-methyluracil (compound 8)]. These manganese complexes, which represent some of the most stable intermediate manganese species observed thus far in the oxidation of carbon-carbon double bonds, show absorption maxima in the 285-296 nm region (epsilon max approximately 4500). The relative reactivities of 6-methyluracil: uracil: thymine are 1: 23 : 194 and the bimolecular oxidation process is characterized by relatively small deltaH++ values and large negative deltaS++ values.


Fluorescent labeling of DNA in solution with covalently bound acriflavin.

We have prepared a fluorescent derivative of DNA based on the acriflavin-Feulgen histological procedure for staining DNA. Our procedure involved binding acriflavin to DNA in solution by reacting the acriflavin with aldehydes formed on the deoxyribose of DNA by controlled removal of a few percent of the purine bases of the DNA. Partially depurinated DNA was reacted with the acriflavin reagent, and unbound acriflavin was removed by chromatography on Sephadex G-25 eluted with phosphate buffered guanidine -HCl. Such single-stranded depurinated DNA bound 0.36 acriflavin molecules per 100 purine bases per h of depurination. DNA containing one bound acriflavin per 200 bases reassociated at 85% of the value of control DNA. The acriflavin - DNA complex showed new absorption maxima at 466 and 370 nm. The fluorescent product had excitation maxima at 304 and 465 nm and an emission maximum at 502 nm. This labeling procedure should be useful in place of or in addition to radioactive labeling for DNA.


Mechanism of hydroxylamine mutagenesis. Crystal structure and conformation of 1,5-dimethyl-N4-hydroxycytosine.

The crystal structure of the title compound, which is a formal analogue of 5-methyl-N4-hydroxycytosine nucleosides, has been determined by X-ray diffraction. The space group is P2(1)/c with a = 7.368 (2), b = 12.096 (3), c = 9.192 (4) A, beta = 113.94 (3) degrees. Three-dimensional intensity data were collected with a four-circle diffractometer, and the structure was refined by block-diagonal least-squares to R = 0.053. The compound is in the imino form, and the exocyclic N4-OH is located essentially in the plane of the pyrimidine ring, and syn to the ring (N(3). There is an intramolecular hydrogen bond involving the N(3)-H as donor and O(4) as acceptor, viz. N(3)-H(31)----O(4)-H. With this conformation, which probably prevails also in solution, the compound would be unable to participate in normal Watson-Crick base pairing. It is shown that a similar situation may prevail for N4-hydroxycytosine nucleosides. The implications with regard to the molecular mechanism of hydroxylamine mutagenesis, with particular reference to the T-even bacteriophages, are discussed. Analogous considerations are applied to an examination of the possible behaviour of hydroxylamine-modified adenine nucleosides.


Effect of starvation of the distribution of free and membrane-bound ribosomes in rat liver and on the content of phospholipid and glycogen in purified ribosomes.

The distribution of free and membrane-bound ribosomes in liver in response to starvation has not been clearly defined. An investigation has been made of the effects of starvation on the content of DNA, RNA, protein, phospholipid and glycogen in rat liver, on the distribution of free and membrane-bound ribosomes, and on the content of phospholipid and glycogen in free and bound ribosome fractions. The results indicate that starvation can produce up to a 50% reduction in hepatic ribosomes without altering either the fraction of rRNA relative to the total RNA or the distribution of free and membrane-bound ribosomes. In addition, the degree of contamination of isolated ribosomes with membranous material does not fluctuate with changes in the nutritional status of the animal. The results suggest that the relative capacities for protein synthesis among the two ribosome compartments are maintained during the early stages of starvation. Further, co-sedimentation with glycogen is not responsible for the presence of membranous materials in purified ribosomes.


Characterization of DNA polymerase associated with nuclear membrane fractions from uninfected and adenovirus 2-infected KB cells.

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.


Comparison of the temperature sensitivity of protein synthesis by cell-free systems from liver of rat and skate (Raja ocellata).

Studies were undertaken to determine the component(s) responsible for the temperature optimum characteristic of the protein-synthesizing system from skate and rat. 1. The macromolecular constituents of rat and skate liver ribosomes are compared. The number of ribosomal proteins is similar in the two species, although most proteins display different electrophoretic mobilities on polyacrylamide gels. The RNAs from the small subunit of skate and rat have similar sedimentation coefficients; however, the RNA from the large subunit of skate ribosomes appeared to be slight smaller than the comparable RNA from the rat. 2. Ribosomes from either rat or skate were capable of supporting poly(U)dependent polyphenylalanine synthesis with soluble factors from either species. 3. Maximal leucine incorporation directed by endogenous mRNA occurred at 35--40 degrees C with post-mitochondrial supernatant from the rat liver and at 20--30 degrees C with that from skate liver. 4. The characteristic temperature sensitivity of protein synthesis was dependent upon the source of cell sap and independent of the source of ribosomes. 5. Elongation factor 1 from both the rat and skate exhibited maximum activity at approx. 30 degrees C. 6. Phenylalanyl-tRNA synthetase from skate liver showed maximum activity at 30 degrees C while that from rat was maximally active at 37 degrees C. The rat enzyme, however, was active at 0--10 degrees C, at which temperature protein synthesis in the reconstructed rat system is virtually absent. 7. The protein-synthesizing capacity of the reconstituted system at various temperatures was closely correlated with the activity of Elongation factor 2 (translocase). Elongation factor 2 from rat liver displayed an optimum at 30 degrees C and lost all activity below 10 degrees C, while this same factor from skate liver showed an optimum at 20 degrees C and significant activity below 10 degrees C. At this low temperature the reconstituted skate liver system continued to exhibit the ability to synthesize protein. These studies suggest that Elongation factor 2 is the component responsible for determining the temperature at which the protein-synthesizing system displays its characteristic maximum activity.


Excision of ultraviolet and gamma ray products of the 5,6-dihydroxy-dihydrothymine type by nuclear preperations of xeroderma pigmentosum cells.

Monomeric products of the 5,6-dihydroxy-dihydrothymine type are produced in the DNA by both ultraviolet and ionizing radiations. The capacity of nuclear preparations from normal and Xeroderma pigmentosum (XP) cells (Complementation groups A, B, C and D) to excise such products from ultraviolet or gamma-irradiated T7DNA was comparable and was independent of radiation induced strand breaks.


The crystal and molecular structure of the polymeric complex of zinc(II) withcytosine 5'-phosphate: metal bonding to both N(3) and phosphate.

Crystal structure analysis of the zinc(II) complex with cytosine 5'-phosphate, (C9H12N3O8P)Zn(H2O), shows that the complex is two-dimensionally polymeric, and the zinc atom is tetrahedrally coordinated to N(3) of the pyrimidine, to two phosphate oxygen atoms and to one water molecule. The zinc atom also forms weak intramolecular interaction with O(2) of the pyrimidine.


Giant RNA in insects. I. Differential changes during Calliphora development and the role of ecdysone.

The pattern of newly synthesized RNA in the epidermis cells of Calliphora during the larval life to the early pupal stage was analysed by electrophoretic separation of RNA extracts on polyacrylamide gels. During larval life, mainly rRNA was synthesized. The elevation of ecdysone concentration during the synthesis of the new larval cuticle resulted primarily in the stimulation of rRNA synthesis. In the transition period from larva to pupa, the principal RNA synthesized was rapidly labelled giant DNA-like RNA, with an average life span of 30 min. Prior to the change in RNA metabolism at this development stage, the synthesis of all classes of RNA was depressed. The initial increase in hnRNA synthesis occurs at the onset of the wandering stage, shortly after the appearance of a small peak of ecdysone and about two days prior to the main peak of hormone secretion and pupariation. During induced pupariation by ecdysterone, 1 h after hormone treatment, more label was observed in giant hnRNA followed by elevation of rRNA synthesis. Processing and maturation of rRNA also seemed to be stimulated.


Stimulation of cell proliferation in rat liver by alpha-hexachlorocyclohexane or partial hepatectomy and end points during G1 of the inhibitory action of beta-diethyl-aminoethylphenyldiallyl acetate-HC1 (CFT 1201), beta-diethylaminoethyldiphenylpropyl acetate. HC1 (SKF 525-A) and actinomycin D.

The present work was designed to study the nature, sequence and temporal position of some inhibitor-sensitive events of the replicative cycle in rat liver. Hepatocyte proliferation was induced by alpha-hexachlorocyclohexane and by partial hepatectomy; the onset of DNA synthesis and of mitotic activity were determined and used as reference points in the cell cycle. Inhibition of cell proliferation was achieved by CFT 1201, SKF 525-A, and actinomycin D. It was found that the inhibitory action of the three agents ends at the same stage of the replicative cycle, 0--2 h before the G1/S transition, in both alpha-hexachlorocyclohexane-stimulated and regenerating rat liver. It is concluded that the molecular events sensitive to CFT 1201, SKF 525-A or actinomycin D are either identical or temporally closely associated; they do not figure in the metabolic activation of alpha-hexachlorocyclohexane.


Incorporation of L-azetidine-2-carboxylic acid into hemoglobin S in sickle erythrocytes in vitro.

L-Azetidine-2-carboxylic acid, the naturally occurring lower homologue of L-proline, is incorporated into hemoglobin S (sickle hemoglobin) in vitro. Sickle erythrocytes from patients with sickle cell anemia incubated with L-[3H] azetidine-2-carboxylate synthesized radiolabeled hemoglobin which when isolated from cell lysates, co-chromatographed with hemoglobin S on DEAE-cellulose columns. The alpha/beta ratio of azetidine carboxylate incorporation into the globin chains of sickle hemoglobin was 0.94, which is consistent with the presence of four proline residues in each polypeptide chain. Incorporation of azetidine carboxylate into hot trichloroacetic acid-insoluble material in sickle erythrocytes indicated that the homologue was present in the polypeptide backbone of the globin chains of sickle hemoglobin. Amino acid analysis of the hot trichloroacetic acid-insoluble material from sickle erythrocytes which had been incubated with radiolabeled azetidine carboxylate indicated that 75% of the radioactivity could be accounted for as intact homologue while 20% of the radioactivity co-chromatographed with alanine. These results suggest that azetidine carboxylate is incorporated unaltered into hemoglobin S in addition to being metabolized to alanine in sickle erythrocytes prior to incorporation into protein. The kinetics of thermal precipitation of hemoglobin S (oxygen ligand) into which radioactive azetidine carboxylate or radioactive proline had been incorporated in vitro is identical. This observation, together with the behavior of hemoglobin S and the globin chains from hemoglobin S containing azetidine carboxylate during ion-exchange chromatography, indicates that homologue replacement of prolyl residues does not significantly alter the overall charge or stability of the hemoglobin S tetramer. Azetidine carboxylate did not inhibit uptake of radiolabeled proline by sickle erythrocytes suggesting that the homologue does not adversely affect amino acid transport in these cells.


Control of protein synthesis by hemin. Isolation and characterization of a supernatant factor from rabbit reticulocyte lysate.

The regulation of protein synthesis by hemin in rabbit reticulocyte lysates is mediated by a hemin-controlled translational repressor protein (HCR) that inhibits polypeptide chain initiation. The effect of this translational inhibitor can be reversed by a high molecular weight protein in the post-ribosomal supernatant fraction. This supernatant factor has been purified approx. 700-fold. It is as effective in reversing the inhibition of protein synthesis due to an early form of HCR (intermediate HCR) as it is in stimulating protein synthesis in the absence of hemin. It is progressively less effective at reversing the inhibition of protein synthesis due to a late from of HCR (irreversible HCR), double-stranded RNA, and oxidized glutathione. The supernatant factor is chromatographically different from the initiation factor IF-MP, isolated from reticulocyte ribosomes, that can also overcome the inhibitory effect of HCR. The supernatant factor does not require hemin for activity, and its action is somewhat suppressed by a level of hemin that is optimal for protein synthesis.


Different susceptibility of protein synthesis to inhibitors of elongation in cell-free systems from plasma cell tumours and reticulocytes.

Plasma cells and reticulocytes are mammalian cell systems which have specialized in the synthesis of a single protein during their differentiation from one common stem cell. To study whether there is a difference in cell susceptibility at the level of elongation, dose vs. inhibition curves of sparsomycin, cycloheximide and emetine in cell-free systems with S-30 fractions from plasma cell tumours (MOPC 63, MOPC 41, RPC 20, MOPC 104 E), reticulocytes and liver were compared. The experiments revealed: (1) all the selected systems are equally sensitive to sparsomycin; (2) the susceptibility of the reticulocyte systems to cycloheximide and emetine is higher than that of the plasma cell tumours. In the dose range of 1 - 10(-7) --5 - 10(-5) M cycloheximide and 1 - 10(-6)--1 - 10(-4) M emetine the reticulocyte system is preferentially inhibited; (3) the sensitivities of all plasma cell tumours are equal; (4) the liver system is more sensitive to emetine than to cycloheximide; (5) the site of the different susceptibility to these antibiotics could be located on the ribosomes; (6) however, when the extracts of the plasma cell tumours were prepared in the presence of hemin, their susceptibility rises and is like that of reticulocytes. These results show that hemin promotes in a cell-specific manner the sensitivity to some inhibitors of protein synthesis.


Inhibition by 5-thio-D-glucopyranose of protein biosynthesis in vitro in spermatids from rat testis.

The effect of 5-thio-D-glucopyranose (thioglucose) upon protein biosynthesis in vitro was examine in testes from mature rats. Thioglucose in vitro is without demonstrable effect upon incorporation of L-[U-14C]phenylalanine into protein by whole testis but inhibits this incorporation by a purified fraction of immature spermatids (stages 1--8) prepared by centrifugal eluctriation; the inhibition is observed with or without glucose added in vitro and is concentration dependent in the range 1--50 mM. Similar inhibition is observed with three other 14C-labeled amino acids (leucine, lysine and glutamate). Mature spermatids (greater than stage 8) and other heterogenous fractions of testicular cells prepared by the same method also show inhibition by thioglucose of incorporation of phenylalanine into protein so that it is not possible to say that the effect is confined to spermatids although it is most pronounced in these cells. Inhibition of protein synthesis in vitro is also observed when thioglucose was administered in vivo (33 mg/kg body wt./day). This change occurs at the minimal dose observed by other workers to produce arrest of spermatogenesis and hence infertility.


Membrane associated phospholipoproteins of Bacillus lichenformis 749.

The membrane-bound penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein that differs from the hydrophilic exoenzyme in that its polypeptide chain carries an additional 25 residues (mostly hydrophilic) with phosphatidylserine as the NH2-terminus. To determine if other phospholipoproteins are present in the plasma membrane, the penicillinase-inducible strain 749 was grown without inducer in the presence of [2-(3)H] glycerol. Electrophoretic separation of the membrane proteins (after removal of free lipids) showed an association of 3H-activity with certain of the proteins which could not be broken by lipid solvents and strongly denaturing conditions. Pronase digestion of the membrane proteins (after solvent extraction) released phosphatidylserine, thus indicating the covalent linkage of protein and phospholipid. Treatment of the isolated membranes with trypsin solubilized the protein portion of some of the phospholipoproteins (as with penicillinase), but not the 3H-labelled fragment. Penicillinase should be considered as the first observed example of a group of phosphatidylserine-containing proteins present in the plasma membrane of B. licheniformis 749 and 749/C.


Factors affecting the compartmentalization of sodium ion within rabbit ileum in vitro.

(1) Net Na+ loss from rabbit ileum, stripped of its serosal muscle layers, into ice cold choline chloride is consistent with loss from two separate pools (rate constants 0.102 and 0.011 min-1). Since cell K+ is lost with a single rate constant, 0.0062 min-1) and inulin, a good extracellular marker, is lost with a single rate constant 0.082 min-1, it is inferred that the fast rate constant of Na loss characterizes loss from an extracellular pool and the slow constant, loss from an intracellular pool. (2) The [Na+] in the inulin space (extracellular) was calculated to be 180 +/- 13 (S.D.) mequiv. and the [Na+] in the intracellular space 30.4 +/- 4.1 (S.D.) medquiv., this provides evidence that the paracellular spaces are, at least 80 mosmol hypertonic to the external Ringer. (3) There is a saturable galactose-dependent increase in both the intracellular and extracellular [Na+]. Extracellular [Na+] is increased to 236 +/- 22 (S.D.) mequiv. Whilst intracellular [Na+] is increased to 42.6 +/- 8.8 (S.D.) mequiv. when Ringer [galactose] is 10 mM. Galactose-dependent increases in total tissue [Na+] can thus be attributed mainly to the increase in extracellular [Na+]. (4) Extracellular hypertonicity, both in the presence and absence of galactose, is dependent upon the [Na+] of the bathing Ringer. 0.1 mM ouabain abolishes the extracellular hypertonicity. This observed extracellular hypertonicity in normally functioning tissue may provide the driving force for transcellular convective flow of salt, water and sugars.


Bidirectional sodium ion movements via the paracellular and transcellular routes across short-circuited rabbit ileum.

1. It has been confirmed that the agent 2,3,6-triaminopyrimidine decreases Na+ conductance in the paracellular pathway of rabbit ileum. 2. Triaminopyrimidine has been used as a means of measuring transcellular bidirectional Na+ flux, and also, of assessing the contribution of the paracellular pathway to transepithlial Na+ flux. 3. Reduction of Ringer [Na+] to 25 mM or incubation with 0.1 mM ouabain reduces paracellular Na+ permeability. This effect may be due to lateral space collapse. Ringer galactose increases serosa to mucosa Na+ flux by a stimulating reflux through the tight junctions. A proportion of net Na+ flux in control tissues is due to asymmetry generated in the paracellular pathway. It is likely that this passive asymmetry results from an osmotic pressure gradient across the tight-junction. 4. Measurement of the tissue isotope specific activity ratio together with bidirectional transcellular Na+ fluxes allows calculation of the four unidirectional fluxes across the mucosal and serosal boundaries. Values obtained for Na+ entry (J12) and exit (J21) across the mucosal boundary are 7.97 alnd 7.13 mumol-cm(-2)-h(-1) respectively. Entry flux (J12) is a saturable function of Ringer [Na+]. The calculated Km is 295 mM and the V is 17.6 mumul-cm(-2)-h(-1). Na+ entry flux is insensitive to ouabain (0.1 mM). Ouabain results in elevation of exit (J21) flux of Na+ across the brush border. D-Galactose causes a saturable increase in Na+ flux (J12) across the mucosal boundary; the Km for this relationship is 1.2 mM and the V 2.17 mumol-cm(-2)-h(-1). The stoichiometry between sugar and Na+ entry is applixmately 1:1. In contrast to the effect of galactose on entry flux, no change in Na+ efflux across the mucosal boundary is observed when Ringer [galactose] is raised. This finding is dissonant with the prediction of the Na+ -gradient hypothesis. The calculated values of exit (J23) and entry (J32) Na+ fluxes across the serosal border are 16.74 and 15.90 mumol-cm(-2)-h(-1). 0.1 mM ouabain markedly reduces both these unidirectional fluxes. This result is consistent with a serosal location of the Na+-pump. Serosal Na+ exit flux J23 increases as a hyperbolic function of Ringer [galactose]. A small galactose-dependent decrease in entry (J32) is also observed. 0.1 mM ouabain abolishes these galactose-dependent changes. 5. The present findings together with those in the previous paper are discussed in relation to the convective-diffusion model for sugar transport.


Phospholipid membrane stabilization by dimethylsulfoxide and other inducers of Friend leukemic cell differentiation.

A large number of low molecular weight polar cryoprotective agents have recently been found to induce erythroid differentiation of Friend leukemic cells in vitro. The effect of these agents on membrane fluidity in phospholipid vesicles was studied by determining the solid-to-liquid crystalline phase transition using differential scanning calorimetry. Some of the inducing agents studies were found to raise the normal transition temperature (Tc) by a few degrees. All of these agents were found to produce a separate transition at a much higher temperature. Changes in the head group of the phospholipid, the pH, the presence of divalent cations, and the addition of other membrane-active compounds were found to significantly influence the inducing agent's effects on the Tc of phospholipid membranes. The ability of the different agents to produce a new transition at a high temperature was found to correlate well with their ability to induce Friend leukemic cell differentiation. The possible mechansims of action of the chemical inducers, and the significance of the observed membrane effects on differentiation and malignancy are discussed. It is concluded that inducing agents decrease the fluidity and stabilize phospholipid membranes, and that their effects in cell differentiation might be initiated by a similar change in the properties of cell membranes.


Characteristics of changes in the intracellular potential associated with transport of neutral, dibasic and acidic amino acids in Triturus proximal tubule.

(1)Introduction of L-alanine and L-lysine into the lumen of the proximal tubule of Triturus kidney evoked an immediate and sustained depolarization of the peritubular membrane potential (Epm) and a small increase in the transtubular potential (Ett). L-Aspartate had no effect. (2) The alanine-induced depolarization was absolutely dependent on the presence of Na+, whereas the lysine-induced one was partially dependent on Na+. In the absence of Na+, alanine usually evoked a transient hyperpolarization of the Epm, while lysine evoked a diffusion potential-like PD change. (3) Addition of alanine or lysine to the peritubular fluid did not cause any immediate change in the Epm, but the cells depolarized with a marked time delay. The delayed depolarization could be ascribed to the entrance of amino acids into the lumen through the nephrostromes and the paracellular pathways. (4) Cellular uptake of alanine and lysine was partially dependent on Na+, while that of aspartate was completely dependent on Na+. (5) Characteristics of the observed electrical events were explained in terms of the differences in the charge transfer associated with transport of these amino acids across the luminal membrane.


Antifolate transport in L1210 leukemia cells. Kinetic evidence for the non-identity of carriers for influx and efflux.

The kinetics of methotrexate transport in L1210 cells are described. Data derived from the measurmenets of initial influx, the complete time-course of uptake, intracellular steady-state level and unidirectional efflux were found to be consistent with a simple empirical equation containing three constants. Properties of the system include the following: (1) saturability of initial influx: (2) approach to steady state during uptake is exponential; (3) the half-time for drug uptake is independent of external concentration and equal to half-time for efflux; and (4) transport is concentrative at low external concentrations, whereas the reverse is true at high external concentrations. These observations are incorporated into a kinetic model which quantitatively accounts for the data on the basis of the hypothesis that influx and efflux take place via different carriers.


Membrane transport properties of bovine articular cartilage.

The thermodynamic parameters which define transport of nonelectrolytes through bovine articular cartilage membranes were evaluated. H3HO, glucose and sucrose were used as permeants. These solutes permeate more readily through the upper layers (near the articular surface) than through the denser deeper layers approaching bone. Cartilage is similar in many respects to a swollen cellulose gel. Viscous-flow contributes importantly to transport within cartilage thus greatly enhancing the movement of nutrients.


Active transport of butyrobetaine and carnitine into isolated liver cells.

1. The liver cells lose the major part of their carnitine during the commonly used isolation procedure by the collagenase-perfusion method. 2. The cells take up carnitine and the carnitine precursor butyrobetaine when these substances are added to the medium. The carnitine content of isolated liver cells can increase to about 15 mM with no apparent harm to the cells. 3. The data indicate the existence of a common carrier in the plasma membrane which mediates the uphill transport of both carnitine and butyrobetaine. The carrier has a high affinity for butyrobetaine (Km=0.5 mM) and a lower one for carnitine (Km=5.6 mM). 4. The intracellular butyrobetaine is hydroxylated to carnitine with a rate of approximately 0.33 mumol-g wet weight-1-h-1 which is sufficient to cover the turn over of carnitine in the whole rat. Carnitine is effectively esterified in the liver cells to acetylcarnitine and long-chain acylcarnitines. 5. Both carnitine and acetylcarnitine are released from the cells. The release of both compounds is probably physiological since it was found that acetylcarnitine constitutes a similar fraction of the total acid soluble carnitine both in the blood and liver of the intact rat.


Identification of the milk fat globule membrane proteins. I. Isolation and partial characterization of glycoprotein B.

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.


Scorpion neurotoxin. Mode of action on neuromuscular junctions and synaptosomes.

Electrophysiological analysis of the effects of scorpion toxin I, one of the neurotoxins from the venom of the scorpion Androctonus australis Hector, upon crayfish neuromuscular junctions has shown that the toxin strongly associates with the nerve terminal to stimulate release of neurotransmitters. The biochemical approach has shown that the binding of scorpion toxin I to rat brain synaptosomes is accompanied by a decrease in their capacity to accumulate gamma-aminobutyric acid. The main effect of the toxin is to stimulate neurotransmitter release. The apparent dissociation constant of the toxin-receptor complex is 0.1-0.2 muM at 22 degrees C. The rate of dissociation is so slow that complex formation seems to be quasi-irreversible. The "quasi-irreversibility" has also been observed in electrophysiological experiments with the crayfish neuromuscular junction. Tetrodotoxin prevents scorpion toxin I action if it is incubated with synaptosomes or with crayfish neuromuscular junctions before scorpion toxin I application. Tetrodotoxin does not reverse scorpion toxin action if it is added to the preparation after scorpion toxin I. Prevention of scorpion toxin action by tetrodotoxin permits measurements of binding characteristics of this toxin to synaptosomes. The dissociation constant of the tetrodotoxin-receptor complex is 2.2 nM at 22 degrees C. No cooperativity is observed in the binding. Because of its high affinity for synaptosomes (and the "quasi-irreversibility" of the binding), scorpion toxin I appears to be a potentially excellent tool for further studies of the molecular mechanism of neurotransmitter secretion.


Phosphorylation in isolated chloroplasts coupled to dichlorophenyldimethylurea-insensitive silicomolybdate reduction.

1. The electron transport in isolated chloroplasts with silicomolybdate as electron acceptor has been reinvestigated. The silicomolybdate reduction has been directly measured as deltaA750 or indirectly as O2 evolution (in the presence or absence of ferricyanide). 2. Silicomolybdate-dependent O2 evolution is inhibited to a similar extent by 3-(3,4-dichlorophenyl) 1, 1-dimethylurea (DCMU) or dibromothymoquinone (DBMIB), indicating the existence of two different sites of silicomolybdate reduction: one before the DCMU block (i.e. at Photosystem II) and one after the DBMIB block (i.e. at Photosystem I). 3. Silicomolybdate-dependent O2 evolution is coupled to ATP synthesis with an ATP/2e- ratio of 1.0 to 1.1. The presence of ferricyanide inhibits this ATP synthesis (ATP/2e- ratio then is about 0.3). 4. Silicomolybdate-dependent O2 evolution is also coupled to ATP-synthesis in the presence of DCMU with an ATP/2e- ratio of 0.6-0.8 characteristic of Site II; in this case the electron transport itself is not affected by uncouplers or energy-transfer inhibitors. 5. The data are interpreted as a further demonstration that the water-splitting reaction is responsible for the conservation of energy at Photosystem II.


Light-induced absorption changes in photosystem I at low temperatures.

Light-induced absorption changes associated with the primary photochemical reaction and dark relaxation in Photosystem I were measured at various low temperatures. A possible temperature-dependent long-range electron tunneling process was suggested to account for the unique temperature dependence of the dark decay process. The kinetics of the light-induced absorption changes are in good agreement with the light-induced EPR changes reported earlier (Ke, B., Sugahara, K., Shaw, E.R., Hansen, R. E., Hamilton, W. D. and Beinert, H. (1974) Biochim, Biophys. Acta 368, 401--408) for the same Photosystem I subchloroplast fragment at comparable temperatures. All absorption changes between 400 and 725 nm at 86degreesK have identical kinetics. The light-minus-dark difference spectrum is very similar to that of P-700 at room temperature, with an additional prominent positive change at 690 nm. Possible contributions by P-430 to the blue and red spectral changes were discussed. It was demonstrated that the intensity of the measuring beam has a drastic effect on the light-induced absorption changes of Photosystem I at low temperatures. Various pretreatments of the Photosystem I fragments such as those that photochemically (or chemically) oxidize the primary donor or photoreduce the primary acceptor abolish the subsequent photochemical reaction. Continuous illumination of the Photosystem I fragments before and during freezing has the same effect. In the temperature range of --20 to --60degreesC, an unusual counter absorption change as well as a counter EPR change were observed.


Positional distribution of fatty acids in rabbit lung phospholipids and triacylglycerols and effect of prolonged hyperoxy.

Investigations have been carried out for the determination of the effect of high oxygen concentration in inspired gas mixutre on the positional distribution of fatty acids in rabbit lung phospholipids and triacylglycerols. The following results were obtained: 1. In the phosphatidylcholine fraction, the high oxygen concentration caused a quantitative increase of palmitic acid (16 : 0) at the alpha-position, and of myristic (14 : 0), heptadecenic (17 : 1) and arachidonic (20 : 4) acids at the beta-position. 2. In t-e phosphatidylethanolamine fraction, the high oxygen concentration caused an increase of oleic acid (18 : 1) at the alpha-position, and of palmitoleic (16 : 1) and heptadecenic (17 : 1) acids at the beta-position. 3. In the triacylglycerol fraction such changes were not observed. In connection with these effects of oxygen, its possible influence on membrane structures in the lung has been discussed.


How valine deprivation and its reversal affect fatty acid metabolism in HeLa cells.

A protocol based on radio gas chromatography demonstrates that when HeLa cells are deprived of valine for short periods of time (6-7.5 h), their overall fatty acid biosynthetic activity is depressed after a latency of a few hours. The transfer of newly synthesized fatty acyl units to phospholipids is curtailed much faster than their transfer to triacyglycerols. Despite the cut-back in fatty acid biosynthesis, valine deprivation causes a lipid accumulation in the cells. Valine deprivation appears to affect de novo synthesis of fatty acid units from acetate more rapidly than desaturation and elongation. When valine is returned to the valine-deprived culture, overall fatty acid biosynthesis is resumed well within 2 h. Newly synthesized fatty acyl units are transferred to both the phospholipids and the 1,2-diacylglycerols of the cells but not initially to the triacylglycerols.


Impaired ganglioside synthesis in rat liver after D-galactosamine administration in vivo.

D-Galactosamine reduces the hepatic content of uridine phosphates, UDP-galactose, and UDPglucose due to an accumulation of UDP-amino sugars; this deficiency can result in severe hepatocellular damage. Alterations of glycosphingolipid synthesis in the early phase of this pathogenic process were studied by measurements of the incorporation of labeled galactose into glycosphingolipids of rat liver. [1-14C]Galactose was injected 2 h after galactosamine administration and the specific radioactivities of the glycosphingolipid precursors, UCPgalactose and UDPglucose, were determined. The specific radioactivity of UDPgalactose, when integrated over the whole period of radioactive synthesis, was four times higher in the galactosamine-treated animals than in the controls; the corresponding ratio of UDPglucose was 0.85. The pattern of the glycosphingolipids isolated from the livers of normal and galactosamine-treated rats resembled that described by Siddiqui and Hakomori (1970, Cancer Res. 30, 2930-2936); GL1, GM3,GM1, GD1, and a small amount of GT could be characterized. The specific radioactivities of glucose and galactose obtained from individual glycosphingolipids were determined in normal and galactosamine-treated livers. The synthesis of the glycosphingolipids was calculated using the respective data of the UDPhexoses. The labeling of glucosylceramide (GL1) was not altered and only a small change of GM3 could be detected; the synthesis of gangliosides GM1 and GD1, however, was inhibited by 95% or more between 4 and 6 h after galactosamine administration.


Feed-back control of cholesterol synthesis in partially hepatectomized rats.

Partial hepatectomy caused a marked stimulation of cholesterol and fatty acid syntheses without affecting serum total cholesterol, total phospholipid and triacylglycerol concentrations of rats so far examined 48 h after the operation. Serum free cholesterol level, however, was increased by the treatment and the ratio of lysophosphatidylcholine to phosphatidylcholine was concomitantly decreased, suggesting the impairment of serum lecithin: cholesterol acyltransferase activity. The lipid content in the liver, especially triacylglycerol and ester cholesterol, was increased markedly by the operation. Feeding of a high cholesterol diet which elevated serum cholesterol and phospholipid levels to the partially hepatectomized rats, accelerated the accumulation of hepatic triacylglycerol and ester cholesterol by the partial hepatectomy. The weight of the regenerating liver was not influenced by cholesterol feeding, which suggested that cholesterol feeding did not inhibit the regeneration mechanism of the liver. The increase of cholesterol synthesis after partial hepatectomy was inhibited by cholesterol feeding. Therefore, it is conceivable that the negative feed-back control of cholesterol synthesis is induced by cholesterol feeding under the stimulated cell divisions of the liver after partial hepatectomy. It is suggested from the present data that a large amount of the cholesterol which is necessary for cell growth can be taken up from serum, when serum cholesterol concentration is high.


Partial purification and properties of the fatty acid elongation systems in the outer and inner membranes of beef liver mitochondria.

Purified outer membrane of beef liver mitochondria was found to elongate medium chain fatty acyl-CoA primer by the incorporation of [1-14C]acetyl-CoA. This enzymic activity, extracted by Triton X-100, was purified 8-fold by ammonium sulfate fractionation followed by chromatography on a Sephadex column. Purified inner membrane, when processed through an identical purification procedure, yielded a second enzyme system which incorporated [1-14C]acetyl-CoA into long chain fatty acids in the presence of medium chain fatty acyl-CoA primer. This enzyme preparation was about four times as active as the preparation from the outer membrane, and used NADH as the reductant for the synthesis. The molecular weights of the inner and the outer membrane enzyme systems, estimated by gel filtration as well as sucrose density gradient centrifugation, were approx. 57 000 and 126 000, respectively. The partially purified outer membrane enzyme system required NADH and a medium chain acyl-CoA primer for the incorporation of [1-14C]acetyl-CoA into long chain fatty acids. KNC stimulated the reaction. NADPH could  substitute for NADH only to a limited extent. Malonyl-CoA was ineffective as a substrate in this reaction. The optimum pH of the reaction was 7.2-7.6 in 0.1 M potassium phosphate buffer. Dithiothreitol, beta-mercaptoethanol, N-ethylmaleimide and high concentrations of ATP and acyl-CoA primer inhibited the reaction. The specificity for the acyl-CoA primer in the reaction was very broad. All the primers tested, C8 to C16, incorporated acetyl-CoA significantly. However, maximum incorporation was observed with dodecanoyl-CoA. Decanoyl-CoA was the best primer for the enzyme system isolated from the inner membrane. About 42% of the radioactivity in the fatty acids synthesized by the outer membrane enzyme system, from myristoyl-CoA and [1-C14]acetyl-CoA, was in palmitic acid. Of the remaining activity, 41% was in stearic acid and about 38% in longer-chain acids. Hence, the elongation of the primer fatty acid by one C2 unit appeared to be the predominant process in this synthesis. In the elongation of myristoyl-C0A by the inner membrane enzyme system, palmitic acid which constituted nearly 78% of the fatty acids synthesized, was the primary product.


[Cathepsin D from horse spleen. I. Purification and study of certain physicochemical properties].

Horse spleen cathepsin D (3.4.23.5.) was purified from crude extract by sodium chloride and ethanol precipitation, column chromatography fractionation on DEAE cellulose and CM Sephadex, re-chromatography on DEAE cellulose and gel filtration. The enzyme has been purified about 3.000 folds with a yield of 30 per cent. The purified enzyme seems to be homogeneous on Sephadex G100, one protein band is apparent on disc electrophoresis. Determined by dansylation the N-terminal amino acid is glycine. A molecular weight of 42,500 +/- 3,000 was obtained with Sephadex G100 gel filtration and light scattering measurements. Amino acid analysis and chemical determinations were performed: cathepsin D is a glycoprotein (2 or 3 osamine residues) including 344 amino acids and 4 disulfide bonds. Spectrophotometric data show that E1cm/1 mg/ml = 1.01 at lambda = 280 nm. ORD measurements indicate about 20 per cent of helicoidal content in the molecule.


Ultrastructure of the atypic muscles associated with terminalial inversion in male Aedes aegypti (L).

Two sets of opposed, crossed muscles are present in the rotating region of the abdomen in male A. aegypti. These muscles undergo changes during rotation of the genitalia that suggest they function as the driving force for rotation. During this rotation, one muscle of each set contracts and the opposed one becomes elongated. The contracting muscles are atypic physiologically. They contract from 300 mum to about 69 mum, and this requires a period of 18 to 24 hours. They shorten only once and those muscle fibers still present after the completion of rotation remain in a contracted condition at least for two weeks. The elongated muscles never shorten; they become stretched to approximately 2.5 times their original length and disappear soon after rotation is completed.


Permeability of trout erythrocytes to nonelectrolytes.

Using a densimeter technique, a kinetic analysis was made, employing both entrance and exit studies, of the permeability of erythrocytes of brook trout (Salvelinus fontinalis), rainbow trout (Salmo gairdneri), German brown trout (Salmo trutta) and cutthroat trout (Salmo clarki) to glycerol, ethylene glycol, thiourea and urea. All of the data indicate that these four nonelectrolytes cross the membrane of the erythrocytes of these four species of fishes by simple diffusion only.


Histochemical changes in gonadal nutrient reserves correlated with nutrition in the sea stars, Pisaster ochraceus and Patiria miniata.

1. Pisaster ochraceus has an annual reproductive cycle in which the gonads increase in size rapidly from October or November and reach a maximum in March to May, after which spawning occurs. In Patiria miniata the gonadal cycle is less pronounced and less regular. In the course of this study, animals spawned in July 1965, May 1966, and May 1967. 2. Histochemical techniques indicate that in both species glycogen or a glycogen-like carbohydrate occurs in the germinal epithelium and the follicle cells of the female, and in the spermatogonia and primary spermatocytes. A storage carbohydrate which is not removed by diastase or amylase is abundant in oocytes in the form of yolk granules 0.5 to 1.5 mu in diameter. 3. Neutral lipid droplets and phospholipid granules are abundant in all oocytes but the smallest. In the testes, lipid droplets are seen only after prolonged starvation. 4. In both species prolonged starvation results in failure of the gonads to achieve their normal size increase. Such gametes as are seen in starving speciments appear histochemically normal in some instances; in other cases they seem to be undergoing breakdown. 5. The histochemical results concerning nutrient reserves of the gonads are generally in agreement with the biochemical findings of earlier workers.


[Conformational heterogeneity of tRNA, detected in the reaction of guanine bases with ketoxal].

The study of kinetic characteristics of the reaction of tRNA guanine bases with kethoxal has shown that temperature, ionic strength and Mg2+ ions, i.e. factors directly affecting the spatial structure of tRNA, influence also on its internal modification. The modification degree under stabilized spatial tRNA structure depends also on the concentration of kethoxal and is expressed in fractional values of the number of modified guanosine residues per tRNA molecule, which indicates the heterogeneity of tRNA for the modification degree. Chromatography of tRNA1 Val preparation on BD cellulose after the exhaustive modification with kethoxal under conditions of stabilized spatial structure has revealed a fraction of molecules completely resistant to the modificator, and a fraction containing differently modified tRNA molecules. tRNA heterogeneity after the reaction with kethoxal (the presence of resistant and reactive forms) indicates conformational heterogeneity of tRNA, expressed in the simultaneous presence of at least two conformer families.


[Structure of electrochemically reduced adenine nucleotides. Their interaction with inorganic phosphate].

The stable reduced form of adenosine phosphates has probably cyclic or dimeric structure of the type: adenine "head"--phosphate "end". Inorganic phosphate produce a stabilyzing effect on reduced adenosine due to a strong ionic interaction with the amino group. In the case of reduced ADP, inorganic phosphate can incorporate into adenine nucleotide, which oxidation results in the formation of small amounts of ATP. A scheme of possible mechanism of this process is given.


[Heterogeneity of crystalline antithyroid phytoprecipitin].

The crystalline preparation of antithyroid phytoprecipitin has been resolved by chromatography on Whatman CM-32 cellulose on a preparative scale into two components, designed respectively A and B. Each component was further resolved into consisting polypeptide chains alpha (mol. weight 7.000) and beta (mol. weight 17.000) by gel filtration on sephadex G-50 in 1 M acetic acid 6 M urea. Homologous chains were comparatively studied by electrophoresis in acrylamide gel, amino acid analysis and peptide mapping technique. Electrophoresis in acrylamide gel with 6 M urea according to Takayama [8] revealed the identical mobility of beta chains in both components A and B, while alpha chains differed. alphaA chain was more basic, than alphaB, i.e. it had greater positive sharge. The amino acid analysis and peptide mapping showed that alphaA chain had one residue of lysine more than alphaB chain. The comparison of beta chains by peptid mapping confirmed their complete identity.


[Localization of pyruvic acid in the histidine decarboxylase of Micrococcus sp. n].

A fragment containing the pyruvic acid residue is isolated from hymotryptic hydrolyzate of alpha-polypeptide chain of carboxymethylated histidine decarboxylase (HD) 14C-phenylhydrasone. The pyruvate residue is estimated to be bind with alpha-amino group of N-terminal phenylalanine in alpha-polypeptide chain. N-terminal alanine is found in HD alpha-polypeptide chain after its reductive amination. It makes possible to determine the estimation of N-terminal amino acid sequence in HD alpha-chain.


[Comparative study of myosin and DTNB-treated myosin with regard to ATP activity and fluorescence].

When studying enzymic and fluorescence properties of myosin and DTNB-treated myosin in the presence of K+, Na+, Li+, NH4+, Ca2+ and Mg2+ cations the following results were obtained. By the intrinsic protein fluorescence techniques no essential structural changes of myosin molecule at the dissociation of the DTNB light chain and activation myosin ATPase in the presence of different cations were found. The decrease of K+-EDTA-, the increase of Mg2+-activated and the stability of Ca2+-activated myosin ATPase may be the result of the modification of SH1 or SH2 sulfhydryl groups when treating the DTNB myosin in our conditions. The different level of decrease of the K+- and NH4+-activated myosin. ATPase may be explained by the fact, that myosin sulfhydryl groups have different effects on the activation of its ATPase by these cations.


[Separation of leghemoglobin from the nodules of lupine, Lupinus luteus L., into components].

The crude leghaemoglobin suspension from root nodules of yellow lupine (Lupinus luteus L.) was separated into five fractions: I, IIa and IIb, IIIa and IIIb using the chromatography on DEAE-cellulose (numbered following the elution order). The visible absorption spectra show that fractions I, IIa and IIIa are met-leghaemoglobins while IIb and IIIb are oxy-forms. IIb and IIIb were converted to IIa and IIIa respectively when oxidized with potassium ferricyanide. The determination of two first N-terminal amino acids confirms the identity of pairs IIa-IIb and IIIa-IIIb. Three components of leghaemoglobin were obtained having the following N-terminal amino acids: Gly-Val- (the first), Gly-Ala- (the second), Ala-Val- (the third). The molecular weights obtained by Archibald's method are 17 900, 17 600, 18 800 respectively. The second leghaemoglobin species which contains more than 50% of the starting protein mixture was used to grow crystals for X-ray study of the tertiary structure of this protein.


[Pathways of corticosteroid biosynthesis in human adrenals (Itsenko-Cushing disease)].

Pregnenolone and its hydroxyl derivatives are of dominant importance in cortisol and corticosterone synthesis in human adrenals (hyperplasic glands and adenomes) removed in Itsenko-Kushing disease. Progesterone is produced in minimal amounts and is nearly not used for steroidogenesis. Participation of 17,21-dihydroxypregnenolone in cortisol synthesis is demonstrated. 17-hydroxyprogesterone is found not to take an important part as cortisol precursor. 21-hydroxypregnenolone is an essential intermediate in corticosterone synthesis. 11-hydroxyprogesterone does not participate in corticosteroids formation, since the rate of progesterone 11-hydroxylation in human adrenal mitochondria is minimal.


[Isolation of cytochrome b556 from the membranes of Micrococcus lysodeikticus bacteria].

Proteins and cytochrome b556 were solubilized from Micrococcus lysodeikticus membranes using Triton X-100 treatment. Passing of this preparation through DEAE cellulose column in the presence of Triton X-100 made possible to isolate cytochrome b556 from other membrane cytochromes and to purify it up to the content of 2.3 nmol per mg of protein. The prostetic group of cytochrome b556 is determined to be protoheme for the spectrum of alkaline pyridinehemochrome.


[Effect of univalent cations on the glutamate dehydrogenase of chlorella].

Effect of univalent cations (Li+, K+, Na+ and Cs+) on the activity and some kinetic properties of the constitutive and the inducible glutamate dehydrogenases (GDH) of Chlorella pyrenoidosa Pringsheim 82T has been studied. All the cations used activate the inducible GDH and produced no such effect on the constitutive GDH. From the analysis of the kinetic behaviour in the presence of K+ the conclusion was made that K+ promotes and stabilyzes a catalitically advantagenous conformation of the inducible GDH. This phenomenon appears to have a physiological meaning, because of a higher K+ concentration in Chlorella cells (about 0.1 M) and its important role in metabolism.


[Antagonism in the action of hydrocortisone and insulin in vivo on enzymes of pyruvate and malate metabolism in adipose tissue].

Alloxan diabetes and injections of hydrocortisone into intact animals for 5-7 days resulted in a sharp decrease of NADP-dependent malate dehydrogenase in rat epididymal adipose tissue. Combined injection of insulin and hydrocortisone did not produce the decrease of the enzyme activity. Insulin injections into alloxan diabetic rats recovered the activity of NADP-malate dehydrogenase up to the control. Pyruvate kinase activity was decreased under diabetes, and insulin injections produced further decrease of the enzyme activity in diabetic rats. Activities of lactate dehydrogenase and NAD-dependent malate dehydrogenase were less decreased under diabetes. Comparison of lactate dehydrogenase isoenzymes spectrum in adipose tissue of normal and diabetic rats revealed a considerable increase of LDH-1 and a decrease of LDH-4 under diabetes. Insulin injections greatly normalized LDH isoenzyme spectrum.


[Frequency distribution of pyrimidine isopliths in the DNA of actinophage F448 and Actinomyces coelicolor 66].

Base composition distribution of pyrimidine isopliths with general formula PyrnPn+1 in DNA of Actinomyces coelicolor 66 Kras. and in DNK of its actinophage F448 were investigater. These DNAs differ both pyrimidine isoplith distribution and in composition. The quality of shorter pyrimidine tracts in Actinomyces coelicolor DNA is higher compared with that in actinophage F448 DNA. The possibility of exogenic origin of actinophage F448 is discussed.


[Extraction and properties of microcapsulated alpha-chymotrypsin].

A method of microencapsulating of the proteolytic enzyme alpha-chymotrypsin into semi-permeable nylon membranes is worked out. The membrane is a polimer of 1,6-hexamethylenediamine and sebacoyl chloride. alpha-Chymotrypsin is enclosed into the capsule together with polyethyleneimine, capable of joining the walls of microcapsules and making the membrane more stable. The optimal concentrations of polyenthyleneimine and alpha-chymotrypsin are 5% and 1% correspondingly. The highest yield of microencapsulated enzyme was obtained for completely acetylated delta-chymotrypsin. The kinetic properties of microencapsulated alpha-chymotrypsin change very slightly as compared to those of the native one.


[Isoelectric focusing of plasma membrane proteins from Strongylocentrotus intermedius sea urchin embryo cells].

14C-amino acids were introduced into the developing sea urchin embryos S. intermedius in 7,5 hours after the fertilization (middle blastula stage). In 30 min the embryos development were terminated, the plasma membrane was isolated and 14C-labelled proteins were recovered from the membrane with 8 M urea and separated by isoelectrofocusing in pH gradient 3,5-10 in presence of 6 M urea. All 14C-label was introduced into two proteins with pI 4,85-4,90 and 5,20-5,25 and the proteins were effectively separated. The minimal molecular weight of the proteins estimated by SDS-acrylamide gel-electrophoresis was 10 000 and 15 000 daltons, respectively.


Amino acid uptake by fetal kidney slices.

Kidney slices of 7-17-week-old human fetuses were incubated for 90 min or less in Krebs-Ringer solution containing aminoisobutyric acid (AIB). Cell to medium ratios of AIB decrease with increasing fetal age. Uptake rate is slower than for mature human tissue. Both water content and extracellular space is high in embryonic tissue as compared to adult.


A survey of X chromatin in the newborn.

Buccal smears were analyzed for X chromatin in 1,000 newborn infants. The mean percentage of X chromatin in the 506 female infants was 39.5 (range 19-61) and in the 494 males 0.13 (range 0-3.0). Evaluation of the data comparing the percentage of X chromatin with caucasian and noncaucasian infants, the weight of the newborn, and the gestation time, indicated no significant differences within the male or female populations. A longitudinal study on 25 female infants for the first 4 days postpartum indicated a significant increase in the percentage of X chromatin in females.


Blood lipids during normal and early weaning in rats.

In young rats reared with their mothers, a gradual increase of serum cholesterol, triglycerides, phospholipids and total lipids was found between the 7th and the 18th to 22nd postnatal day. Then the concentrations of lipids gradually decrease. Early weaning at the 18th postnatal day causes a precocious decrease of the concentration of all studied lipids in blood so that their values on days 22 and 26 are significantly lower than in animals weaned on the 30th postnatal day. The body weight of early weaned rats decreases only slightly and returns to normal by the 30th day. The changes in serum lipids after early weaning are caused by differences in the amount of cholesterol and other lipids in breast milk, in comparison with those in the standard laboratory diet. Perhaps they may participate in disturbing the homeostatic equilibrium of cholesterol in early weaned rats.


Lack of correlation between clinical data and growth hormone concentrations in cord blood.

Growth hormone concentrations were measured in 182 umbilical cord blood samples taken at the time of birth. The mean value in 165 full-term, normal weight infants was 31.5 +/- 25.9 ng/ml SD. 14 prematures had significantly higher levels (50.0 +/- 38.4 p less than 0.01). There were no statistically significant correlations to any of the following parameters: complication of pregnancy by toxaemia, duration of labour, presence of umbilical cord contortion, perinatal distress, Apgar index, mode of delivery, body weight, body length, ratio of weight to length, and blood glucose. There was a wide scatter of individual values in this study as well as in previous investigations. We think it unlikely that those variations are purely accidental. Hence, yet undefined factors must play a major role in the regulation of GH secretion in the perinatal period.


Adenylate cyclase activity in human tissues.

Adenylate cyclase activity in liver, lung, pancreas, and heart ventricle, from full-term and premature newborns has been assayed. Of all tissues studied, heart had the highest activity. Fluoride 10 mM stimulates adenylate cyclase in all tissues. Glucagon evoked a small activation of liver adenylate cyclase.


A study of the permeability of the guinea pig placenta to citrate using recirculating placental perfusion technique.

The role of the guinea pig placenta in fetal citrate metabolism has been studied by perfusing the placenta in situ. The initial citrate concentrations in the perfusate plasma were lower than in the maternal plasma but rose progressively until, in some cases, they exceeded those in the mother. The rise was uninfluenced by intravenous administration of citrate to the mother. Citrate therefore does not enter the perfusate by simple diffusion from the maternal compartment. It may be synthesised within the placenta. From the rate of accumulation of citrate in the perfusate, the supply to the fetus was calculated to be small compared to the fetal metabolic rate. Citrate added to the perfusate became distributed in a space similar to the extracellular space and no change occurred in maternal citrate concentrations. Thus, the placenta does not appear to metabolise citrate or allow transport to the mother.


Effect of diet on renal response to salt challenge in neonatal piglets.

In piglets 14-40 days of age, blood pressure and glomerular filtration rate increased significantly with age and were lower than those reported for adult pigs. There was no effect of dietary salt on the maturation of these parameters and on tubular sodium reabsorption, although the piglets on a low salt diet had lower inulin space than piglets receiving higher dietary salt. After the completion of a 24-hour salt challenge there was little evidence of sodium retention and there was no effect of previous dietary salt intake on sodium excretion. However, salt challenge resulted in higher blood pressure and elevated serum sodium only in piglets on a low sodium diet. These results suggest that in contrast to puppies and infants, the renal response to salt challenge is physiologically adequate in piglets at 14-40 days although glomerular function is still immature. Dietary salt history appears to have little effect on the degree of sodium excretion during salt challenge in this age range.


Indication of increase of the lecithin/sphingomyelin (L/S) ratio in lung fluid of lambs maternally treated with metabolite VIII of Bisolvon.

Metabolite VIII of Bisolvon (Met. VIII) was administered to six pregnant ewes (i.m. daily 5 or 10 mg/kg for 7-9 days). The treatment was started at the 116th/117th or 117th/118th day of gestation and in one case the drug was given to the fetus (i.v. 2 mg/ml/h). Fetal lung fluid obtained from chronically implanted catheters was used for the estimation of the lecithin/sphingomyelin ratio. The maternal treatment with Met. VIII resulted in a significantly increased growth of the L/S ratio compared to the control group (n=4). In three lambs a sharp increase of the L/S ratio was noticed during the last days of Met. VIII treatment. The time gap between first treatment and noticeable effect was 6-8 days. It is postulated that the noticed increase of L/S ratio is the consequence of Met. VIII treatment and very unlikely the result of a release of glucocorticoticoids.


Influence of oxytocin and meperidine on the isolated human umbilical artery.

The influence of oxytocin and meperidine on responses of the isolated umbilical artery to acetylcholine (ACh) and/or 5-hydroxytryptamine (5-HT) was investigated. 32 preparations from 21 normal full-term deliveries were utilized. The Bunce double-blade hemostat provided an excellent means to collect cord specimens with the least postpartal changes. A method of simultaneously measuring the resistance to flow and longitudinal tension in the perfused artery was found to be a sensitive way to register functional responses. Results showed that oxytocin enhanced the response of the umbilical artery to ACh and 5-HT while meperidine blocked the constriction produced by 5-HT. Both these effects lasted from 30 min to more than 2 h and were reversible.


Liquid ventilation in immature newborn rabbits.

Rabbit fetuses delivered 3 days before term were artificially ventilated with air, oxygen, or oxygenated liquid fluorocarbon for 1.5-3 h. Oxygenation, as revealed by skin colour and ECG was superior in liquid-ventilated animals. Also, liquid ventilation was less harmful to the bronchiolar epithelium and, in contrast to ventilation with gas or oxygen, did not cause the formation of hyaline membranes.


Heterogeneity of pituitary and endogenous plasma human growth hormone from fetuses, premature and full-term newborns.

Fetal and adult human growth hormone (HGH) extract from human pituitaries as well as plasma from premature and full-term newborns and children were submitted to chromatography on Sephadex G-75 and the immunoreactive HGH (IRHGH) in the elution fractions was determined quantitatively by radioimmunoassay. Three fractions of IRHGH could be differentiated according to the elution pattern: 'big-big', 'big' and 'little' IRHGH. In fetal pituitary extract only 'big-big' and 'little' IRHGH were found, whereas in adult pituitary extract all three forms of IRHGH were present. In the plasma samples studied no 'big-big' form was found; only 'big' and 'little' IRHGH appeared. In addition, plasma samples of premature newborns had a smaller proportion of the 'big' IRHGH than did those of the full-term newborns and older children.


Precursor and end product effects upon indoleamine maturation in mouse brain.

The effects of tryptophan loading and the acid transport inhibitor, probenecid, on the maturation of 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) have been studied in the maturing mouse brain. Tryptophan loading elevates already high endogenous tryptophan at all stages studied. It does not alter 5-HT or 5-HIAA at day 1 but does elevate levels of both at later stages. Probenecid does not affect 5-HT at any age or 5-HIAA at day 1, but does cause increasingly greater elevations of 5-HIAA at later stages. The results indicate that tryptophan-5-hydroxylase is substrate saturated in early postnatal stages but not later, and that the early postnatal brain lacks an acid transport mechanism for 5-HIAA egress but develops this at later stages.


Thyroid hormone and cell formation in the developing rat cerebellum.

Effects of neonatal hyperthyroidism on cell formation in the developing rat cerebellum were reinvestigated. Administration at birth of excessive doses of thyroxine or triiodothyronine led to an early stimulation of cell acquisition, followed by a permanent deficit of cells in the cerebellum. The corrective effects of physiological doses of thyroxine on the troubles of the histological and biochemical development of the cerebellum in thyroid-deficient animals were also studied. As early as 6 days, cell maturation and formation were already retarded in animals treated with propylthiouracil, but, as previously reported, cell formation was prolonged and the final number of cells was normal. Administration to thyroid-deficient animals of progressively increasing doses of thyroxine, nearly equal to the amounts of hormone secreted by the thyroid gland of the developing normal rat, returned the evolution of the cerebellar wet weight and of the cerebellar DNA to normal, as well as the histological maturation of the cerebellum, even if it did not entirely correct the retardation of body growth. These results are consistent with the view that thyroid hormone early stimulates maturation of the cerebellar germinative cells and subsequently interacts with cell formation in the cerebellum, and that this action is physiological.


Medical devices as medical problems: the regulation of devices for clinical use.

The development of statutes to regulate the safety and efficacy of medical devices has been proceeding rapidly. The protection of the consumer from dangerous and ineffective devices is obviously necessary and must be carried out rapidly and efficiently. The electrical shock hazard in prevalence and violence is a case in point. There is no question that many devices are not well made and not suited for the function for which they are sold. These must be controlled, but at the same time we must be careful not to destroy the private research and development efforts which are producing reliable instrumentation through over control and regulation.


An endoscopic carrier with complete directional control: design principles.

We describe a carrier for instruments used in endoscopy. It consists of a train of articulated segments whose angular displacement is controlled by a series of magnetic clutches. The operator guides the leading segment under view from the carrier's proximal end, and the guide angle is repeated automatically at the articulations of all following segments. This mechanism is used to lead the entire length of the carrier around curves or flexures. A central channel accepts instruments for observation and manipulation. We believe that endoscopic examinations particularly of the upper and the lower digestive tracts will become much easier to perform with this new endoscopic sonde.


An elbow orthosis.

An elbow orthosis for unilaterally handicapped persons has been developed. The operating principles and construction of the orthosis are described and its important parameters are determined. The procedure of parameter adjustment is given in detail and the area of application is indicated. Preliminary clinical results are discussed.


The measurement of urethral resistance and mural tension using simple programmable modules.

The theory of urethral resistance and the derivation or mural tension have been discussed. A method for the continuous calculation and presentation of these parameters has been described. Urethral resistance and mural tensions are altered by the presence of bladder outflow obstruction and have been shown to return to normality after the obstruction is removed.


A possible new sorbent dialyser for haemodialysis.

A system is proposed for haemodialysis which involves both dialysis and adsorption. The blood compartment is separated by a membrane from a compartment containing adsorbents and ion exchangers under reduced pressure. Urea is broken down by urease adsorbed on an ion exchanger. The ammonia produced is pumped out and the rest of the unwanted substances are bound to the adsorbing materials. The geometry and principles governing the operation of the device obviate the use of circulating dialysate fluid. Preliminary results from in vitro experiments with such a system are reported and the possibility of using this system clinically is discussed.


Patient monitoring in the operating theatre.

Anaesthetised patients are monitored to ensure their safety. Simple clinical observations must not be replaced by electronic instruments--these provide an extension of the clinical senses. The choice of parameters for monitoring is discussed. The design of the Ninewells main operating theatre suite is described. An 8-channel bourne in the base of the theatre table conveys patient signals to a 4-channel recorder in a monitoring laboratory. Outputs are displayed on a wall mounted display in theatre. Two-way speech intercommunication exists with monitoring technician and students.


A prosthetic vas deferens.

Six dogs were implanted with the synthetic vas deferens device as part of our continuing research into the development of a reversible occlusive device for implant in the transected vas deferens. The experimental objectives were to assess the effects of sperm path length and duration of aspermia on total sperm count and motility by implanting three different lengths of the device in animals with different degrees of aspermic duration induced by bilateral vasectomy. Device lengths varied from 1.55 cm to 4.3 cm including the pilot tubes. The prosthesis is fabricated entirely of silicone rubber with Dacron velour tissue ingrowth material bonded to the outside surface of the ring to which vas ends are sutured. The pilot tubes extend 4 mm beyond the suture rings and are 0.6 mm OD with a 0.3 mm ID that extends through the entire length of the device. The flexible pilot tubes are an important feature of this design and insert into the vas lumen ensuring alignment between the device and the vas deferens while maintaining the necessary resiliency to adapt to the inherent movements of the vas without perforation. All animals included in these experiments were carefully selected and conditioned before implant. The evaluation of performance was based on semen samples collected at one week intervals. Sperm subsequently appeared in the ejaculate of all 6 dogs, demonstrating that the restoration of sperm transport can be restored after bilateral vasectomy through the implantation of a prosthetic vas deferens. The potential application of this device to the human male for the restoration of fertility appears excellent.


Concentrating engines and the kidney. III. Canonical mass balance equation for multinephron models of the renal medulla.

The canonical mass balance relation derived for the central core model of the renal medulla is extended to medullary models in which an arbitrary assemblage of renal tubules and vascular capillaries exchange with each other both directly and via the medullary interstitium and in which not all of the vascular loops or loops of Henle extend to the papilla. It is shown that if descending limbs of Henle and descending vasa recta enter the medulla at approximately plasma osmolality, the concentration ratio is given by: r = 1/[1 - ft(1 - fu)(1 - fw)], where ft is fractional solute transport out of ascending Henle's limb, fu is fractional urine flow, and fw is fractional dissipation; fw is a measure of the solute returned to the systemic circulation without its isotonic complement of water. A modified equation that applies to the diluting as well as the concentrating kidney is also derived. By allowing concentrations in interstitium and vascular capillaries to become identical at a given medullary level, conservation relations are derived for a multinephron central core model of the renal medulla.


Lecithin translational diffusion studied by pulsed nuclear magnetic resonance.

The translational diffusion coefficient of egg yolk and dilauroyl lecithin in optically isotropic phases containing sodium cholate has been measured using the pulsed NMR magnetic field gradient method. After a correction for geometrical factors the measured diffusion coefficient is found to agree well with previous determinations in phospholipid systems. The experimental data imply that the cubic mesophase of the lecithin-sodium cholate-water system contains continuous lipid aggregates. A possible model of the arrangement of the different amphiphile molecules in the cubic phase is discussed.


The interaction of the polyene antibiotic lucensomycin with cholesterol in erythrocyte membranes and in model systems. III. Characterization of spectral parameters.

The variations of optical density and fluorescence of lucensomycin are good indices of the binding of this polyenic antibiotic to membranes. The former parameter reflects more generally the binding to any site present in the membrane, while the latter is more specific for binding to cholesterol. The chromophore of the lucensomycin-cholesterol complex has a relatively long lifetime, is almost immobile in the membrane, and is not accessible to water-soluble fluorescence-quenching agents. The stoichiometry, evaluated fluorometrically, corresponds to about two cholesterol molecules per polyene. In colloidal cholesterol suspensions, the extent of binding as a function of free polyene concentration is described by rectangular hyperbolae, the dissociation constant being, however, dependent on the sterol concentration. In erythrocyte membranes, on the other hand, and even more markedly in model systems containing appropriate solvents, the combination between lucensomycin and the sterol sites is described by sigmoid titration curves, indicative of cooperative effects, and probably due to solvation of cholesterol.


Dynamics of fluorescence marker concentration as a probe of mobility.

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.


Effects of removing extracellular Ca2+ on excitation and adaptation in Limulus ventral photoreceptors.

In Limulus ventral photoreceptors, removing extracellular calcium (Ca2+o) increases the median latency of light-evoked discrete waves. Removal greatly lengthens the time-to-peak of responses in the dark-adapted cell, but not in the light-adapted cell. Removal does not block light-adaptation or the light-induced rise in intracellular calcium (Ca2+i). These results are interpreted in terms of the hypothesis that both sensitivity and the kinetics of excitation are dependent on Ca2+i, and that Ca2+i is dependent on Ca2+o in the dark-adapted cell, but in the light is dependent largely on Ca2+ released from intracellular compartments.


Binding isotherms and the interaction between proflavin and a DNA of high G-C content.

Binding isotherms corresponding to several situations of ligand binding to a linear polymer are calculated, including cases of cooperativity or anticooperativity between the bound ligand states, multiple binding modes that are competitive or non competitive, and possible exclusive of an arbitrary number of adjacent sites upon occupancy of a site by a single ligand. The sequence generating function method of Lifson and Bradley is used, requiring the assumption that no end effects are involved. The case of strong binding of the dye proflavin to a DNA of high G-C content, that of M. lysodeikticus, is considered in detail, and a single model capable of reconciling the available kinetic and equilibrium data on this system, involving two competing binding modes, is discussed.


Scanning gel chromatography: determination of transport parameters from dynamic profiles measured at low solute concentration.

Direct optical scanning of solute boundaries in large zone gel chromatography experiments provides an accurate means of determining boundary profile shapes and rates of motion. A method has been developed for correcting such boundaries to a constant time frame, eliminating the distortion which arises from finite column scanning rate. Centroids of the corrected profiles can be used to determine the partition cross section for the solute of interest. The partition cross section and flow rate determine translational motion within the column. The axial dispersion coefficient, L, which characterizes rate of boundary spreading may also be calculated from the profiles. In order to explore these procedures a study of four noninteracting solutes was conducted. Partition cross sections determined from rates of motion of boundary centroids were found to be in good agreement with those determined by the equilibrium saturation method on the same column. In order to explore the lowest concentration limits of the technique and to illustrate the boundary characteristics for a self-associating solute, a study of carboxyhemoglobin was conducted over a wide concentration range. From measurements at 220 nm the lowest concentration where useful data could be obtained was 2 micrograms per ml (0.12muM heme). These results establish validity of the procedures used in analyzing the rates of boundary transport and in studying solute transport over a wide range of conditions.


Active enzyme gel chromatography: II. Computer simulations.

The behavior of an enzyme undergoing reaction while on a gel chromatography column has been studied by computer simulation using the steady state assumtion for a system with a single enzyme-substrate complex. The profiles of the enzyme-substrate complex, product, and substrate were examined varying the parameters of kcat, flow rate, partition coefficient dispersion, and time. These investigations confirm that much information about both the active enzyme and the product may be obtained by examining the product profile alone, varying the power of applying scanning gel chromatography to active enzyme systems.


Determining the base sequence of RNA molecules using continuous degradation kinetics.

A kinetic approach to sequence analysis is presented. A polymer chain composed of a small number of monomer types is degraded continuously with degradation proceeding in only one direction of the chain. Ordinarily, the direct determination of the sequence from the amount of degraded monomers is hampered by the rapid loss of reaction synchrony. However, it is shown, that if the monomers can be measured reasonably accurately, one can solve a system of linear equations with the unknowns giving the positions of the monomers in the chain, and the syn chrony loss due to the stochastic nature of the degradation process can be accounted for. Model calculations are presented.


Rheology of fibrin clots. III. Shear creep and creep recovery of fine ligated and coarse unligated closts.

Creep and creep recovery of human fibrin clots in small shearing deformations have been investigated over a time scale from 24 to 10(4) s. Coarse, unligated clots and fine clots ligated by fibrinoligase in the presence of calcium ions were studied to suppliement previous data on coarse ligated and fine unligated clots. Stress was found to be proportional to strain up to at least a maximum shear strain (in torsion geometry) of 6.2%. The initial modulus (25 s after imposition of stress) is proportional to approximately the 1.5 power of concentration for fine ligated and coarse unligated clots. For fine unligated closts there is comparatively little creep subsequent to the initial deformation; ligation (in this case involving mostly the gamma chains) reduces the creep to nearly zero. For coarse unligated clots, there is substantially more creep under constant stress, and creep recovery is not complete. Ligation (in this case involving both camma and alpha chains) alrgely supresses the creep and causes the recovery to be complete. If the structure if fully formed before creep begins, tests of creep recovery by the Boltzmann superposition principle show adherence to linear visoelastic behavior for all four clot types. Otherwise, the Boltzmann test fails and the recovery is much less than calculated. For fine ligated clots, the observed recovery agrees well with that calculated on the basis of a dual structure model in which an additional independent structure is built up in the deformed state, so that the state of ease after removal of stress is a balance between two structures deformed in opposite senses. It is postulated that the coherence and elastic modulus of the fine ligated clot are largely due to steric blocking of long protofibrils with a high flexural stiffness. In the coarse clot, it is proposed that the structure involves extensive branching of thick bundles of protofibrils, which become permanently secured by the ligation of the alpha chains of the fibrin.


The molecular mechanism of temperature enhancement of proton magnetic relaxation rates in methaemoprotein solutions. III. The effect of sixth ligand in high-spin derivatives.

The distinctions of the solvent-proton longitudinal magnetic relaxation (PMR) mechanisms between high-spin ferric aquo and fluoro complexes of some haemoproteins are discussed here. It becomes apparent that the "transition" from the exchange-limited PMR to the fast-exchange PMR mechanism upon addition of fluoride to some of the aquocomplexes is due to a more intense solvent-dynamics in the vicinity of the paramagnetic haem in the fluoromet derivatives. The can be rationalized by a conformational change induced by the fluoride ion, an effect not observed by X-ray analysis thus far. A possible mechanism of this change is indicated here.


Chemical ionization and high resolution electron impact mass spectra of 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide and three of its metabolites.

The electron impact and the hydrogen and methane chemical ionization mass spectra of 5,6-dihydro-2-methyl-1,4-oxathin-3-carboxanilide, the sulfoxide and sulfone derived therefrom, and 2-(2-hydroxyethylthio)-acetoacetanilide enol have been determined. All four compounds show abundant molecular ions in the electron impact spectra and abundant [MH]+ ions in the methane chemical ionization spectra (along with the expected [M + C2H5]+ and [M + C3H5]+ ions), but relatively low [MH]+ ion signals in the hydrogen chemical ionization spectra. From high resolution mass measurements and metastable transitions determined by metastable ion refocusing, electron impact fragmentation mechanisms have been established. For 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide, 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4-oxide and 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4,4-dioxide the major fragmentation mode involves loss of the anilino radical from [M]+, followed by loss of C2H4. Fragmentation to form the aniline molecular ion increases in importance with increasing oxidation state of the sulfur. In the chemical ionization of these three compounds fragmentation of [MH]+ proceeds in a similar fashion by loss of neutral aniline and by formation of protonated aniline. The electron impact mass spectrum of 2-(2-hydroxyethylthio)acetoacetanilide is dominated by the molecular ion and the aniline molecular ion. However, in the chemical ionization mass spectra characteristic fragment ions involving loss of water and loss of aniline from [MH]+, as well as protonated aniline, are observed and serve to characterize the compound.


Field desorption mass spectrum of glucagon.

A low resolution, high sensitivity study has been made of the field desorption mass spectrum of glucagon, a 29 amino acid residue underivatized peptide. The highest molecular weight fragments observed, when spectra were taken under conditions which minimized surface reactions of the peptide on the emitter, were found to contain no more than 18 amino acid residues. The most probable fragmentation processes for ions larger than m/e 600 were the production of fragments containing 10, 11 or 12 amino acid residues.


Selected ion monitoring assay for the bronchodilator pirbuterol.

A selected ion monitoring assay has been developed to measure plasma levels of the bronchodilator pirbuterol in animals and man. Pirbuterol is extracted from plasma with acetone + potassium carbonate and after further purification is converted to its trimethylacetyl derivative. With the use of the stable isotope labeled internal standard, pirbuterol-d9, precisions of 10% and 6% were achieved at plasma concentrations of 1 and 20 ng ml-1, respectively. The synthesis of the deuterium labeled internal standard is described.


Simultaneous estimation of propoxyphene-do and propoxyphene-d2 levels by quantitative mass fragmentography. Potential utility in multidose investigations.

A quantitative mass fragmentographic method for the simultaneous determination of labeled and unlabeled propoxyphene in plasma is described. Dogs treated daily with propoxyphene-do were treated with a pulse dose of propoxyphene-d2 at day 20. It was found that following an initial rapid equilibrium phase levels of propoxyphene-d2 fell more rapidly than those of propoxyphene-do. This result suggests that 'deep' pools of tissue bound propoxyphene exist which exchange very slowly with drug present in the central compartment. Experimental evidence is presented which demonstrates that the difference in behavior of propoxyphene-do and -d2 is not due to unanticipated secondary isotope effects.


Propoxyphene: pathways of metabolism in man and laboratory animals.

Through the combined use of stable isotope labeling and gas chromatographic mass spectrometric analysis, the metabolic patterns for propoxyphene have been determined in laboratory animals and man. The rat and dog eliminated propoxyphene and its metabolites principally via the bile, while the rabbit more closely resembled man in excreting the metabolic products into urine. Metabolites in rat and rabbit existed as conjugates, whereas in dog and man the metabolites were excreted as a mixture of the free and unconjugated forms. The primary route of metabolism in all species studied was N-demethylation. However, the rat and rabbit extensively hydroxylated propoxyphene and its metabolites prior to elimination. Metabolites arising from ester hydrolysis were found in rat and man. N-acetylated products were identified in all four species. A metabolite formed from cyclization and dehydration of dinorpropoxyphene was isolated in urine and was further identified as a circulating metabolite in dog plasma.


Replacement of a chlorine with a methylsulfonyl group in the metabolism of propachlor (2-chloro-N-isopropylacetanilide).

Urine from rats and sheep given single doses of [14C]propachlor contained 14C metabolites in which the chlorine of propachlor was replaced by a methylsulfonyl group. Methylsulfonyl-containing metabolites were also isolated from the urine of rats given an intraperitoneal dose of the cysteine conjugate of [14C]propachlor; this indicated that the methylsulfonyl-containing metabolites resulted from metabolic reactions subsequent to the mercapturic acid pathway.


The replacement of trideuteromethyl by methyl groups when using methane as a reagent gas in chemical ionization mass spectrometry.

The methane chemical ionization spectra of a number of tertiary and quaternary amines with a N-trideuteromethyl group show substitution of this group by methyl. The isotopic purity of the original amines was shown to be greater than 99% using ammonia as a reagent gas.


Chemical ionization and field desorption mass spectrometry of the gentamicins.

Gentamicin sulphate employed in therapeutics consists of a mixture of three major aminoglycoside components, the gentamicins C1, C1a and C2. In the electron impact mass spectra of these components weak parent ions may be observed but they are of no diagnostic value in the commercial mixture. Chemical ionization and field desorption spectra of individual gentamicin components and commercial mixtures are reported and discussed. Isobutane chemical ionization spectra exhibit some fragmentation, but under optimum field desorption conditions little glycosidic cleavage is observed and [M + H]+ ions dominate the spectra.


Mass spectrometric investigation of peptide amides.

The reaction products of trifluoroacetic anhydride with peptide amides are identified as stable heterocyclic imidazolin-5(4)-ones, which can be analysed by gas chromatography mass spectrometry. The mass spectra show intense molecular ions and allow the assignment of the amino acid sequence.


Field desorption mass spectra of methotrexate and folic acid analogs.

Field desorption mass spectra of methotrexate and folic acid analogs yield abundant molecular or quasimolecular ions. At emitter currents above the best anode temperature, fragment ions which are useful as further confirmations of structures are observed.


The analysis of terbutaline in plasma and urine at therapeutic levels using mass fragmentography.

A sensitive method for the qualitative and semiquantitative determination of terbutaline in plasma and urine has been developed. After a single oral dose of 5 mg terbutaline sulphate the drug could be analysed easily. The detection limit in plasma is less than 1 ng ml-1.


Specific deuteromethylation by the Eschweiler-Clarke reaction. Synthesis of differently labelled variants of trimethylamine and their use for the preparation of labelled choline and acetylcholine.

Reductive methylation (the Eschweiler-Clarke reaction) was used as a route for the synthesis of differently deuterium labelled variants of trimethylamine with the ultimate aim of preparing labelled variants of choline and acetylcholine. Combinations of unlabelled and labelled formaldehyde and formic acid yielded symmetrically labelled trimethylamines of high isotopic purity. The labelled congeners to acetylcholine and choline that were prepared subsequently provide suitable internal standards and tracers to be used in mass spectral analysis and in the evaluation of the pharmacokinetics of the parent compounds.


Hereditary angioedema: modification of clinical manifestations with androgens.

HAE is an autosomal dominant trait of decreased levels or function of circulating and tissue C'1 esterase inhibitor. The clinical illness is characterized by disabling episodes of peripheral, oropharyngeal and gut-wall edema. Long-term fluoxymesterone treatment of 5 affected males (393 patient-months) and oxymetholone treatment of 6 affected females (204 patient-months) has significantly decreased the frequency of attacks of edema without substantive side effects.


Heterogeneity in the campomelic syndromes: long and short bone varieties.

There appears to be definite heterogeneity within the campomelic syndromes. We are broadly classifying these into the classic or long bone campomelic syndromes and 2 distinct short bone varieties designated as the normocephalic short-boned and craniosynostotic short-boned types, respectively (Table 1).


Abnormal hemoglobins in a quarter million people.

Hemolysates of erythrocytes from more than a quarter million people in Alabama were electrophoresed on cellulose acetate, pH 8.4, and those samples exhibiting an abnormality were also electrophoresed in citrate agar, pH 6.0. The globin chains of mutants other than Hb S and C were electrophoresed in urea-mercaptoethanol buffers at both pH 8.9 and pH 6.0, and 60 of them were also analyzed structurally. Of about 6000 samples from whites, only three contained abnormal hemoglobins--Hb D Los Angeles, Hb J Baltimore, and one unidentified. Of 249,000 samples from blacks, about 29,000 contained electrophoretically detectable abnormalities, most of them associated with Hb S or C, present in a frequency of about 9% and 3%, respectively. About 1000 samples resolved into patterns of potential clinical significance. Twenty other mutant hemoglobins were detected, in various genetic combinations in 164 kindreds; four of these-Hb Alabama, Montgomery, Titusville, and Mobile--were previously unknown. The methods used are rapid, economical, and well suited for large scale surveys. They provide highly specific characterizations of many mutant hemoglobins, and no discrepancies were found between the presumptive identifications based on these characterizations and the definitive identifications obtained from structural analyses.