Preparation of membrane vesicles from isolated myelin: studies on functional and structural properties. Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in 22Na+ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myeline basic protein from 0--150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.


Binding of polylysine to charged bilayer membranes: molecular organization of a lipid.peptide complex. The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.


Protein-bound histidine, as well as protein-bound serine, residues are sites of phosphorylation in the synaptic plasma membrane. When synaptic plasma membrane fragments are incubated with ATP in the presence of Mg2+, phosphate is transferred, not only to protein-bound serine, but also to protein-bound histidine. The phosphorylation of protein-bound serine is stimulated by cyclic AMP and has a Km for ATP of about 0.12 mM, both in the presence and absence of cyclic AMP. By contrast, the phosphorylation of protein-bound histidine is unaffected by cyclic AMP and does not follow Michaelis-Menton kinetics since a non-linear double reciprocal plot is given when activity is measured at various ATP concentrations.


Plasma membrane cyclic AMP-dependent protein phosphorylation system in L6 myoblasts. Plasma membranes can be isolated without disruption of cells by the plasma membrane vesiculation technique (Scott, R.E. (1976) Science 194, 743-745). A major advantage of this technique is that it avoids contamination of plasma membranes with intracellular membrane components. Using this method, we prepared plasma membranes from L6 myoblasts grown in tissue culture and studied the characteristics of the protein phosphorylation system. We found that these plasma membrane preparations contain protein kinase which is tightly bound to the membrane and cannot be removed by washing in EDTA or in high ionic strength salt solutions. This protein kinase activity can catalyze the phosphorylation of several exogenous substrates with decreasing efficiency as acceptors of phosphate: calf thymus histones f2b, protamine and caseine. Cyclic AMP causes a dose-dependent stimulation of protein kinase activity; the highest stimulation (4-fold) is achieved at concentration 10(-5) M cyclic AMP. Cyclic AMP-dependent stimulation can be completely inhibited by heat-stable protein kinase inhibitor isolated from rabbit skeletal muscle. On the other hand, cyclic GMP does not affect the activity of protein kinase. Plasma membrane-bound protein kinase also catalyzes the phosphorylation of endogenous membrane protein substrates and this is also stimulated by addition of cyclic AMP. Analysis of plasma membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that specific polypeptides are phosphorylated by cyclic AMP-independent and by cyclic AMP-dependent protein kinase systems. The results of these studies demonstrate the presence of endogenous cyclic AMP-dependent and -independent protein phosphorylating systems (enzyme activity and substrates) in purified plasma membrane preparations. These data provide a basis for further investigations on the role of plasma membrane phosphorylation as a regulator of membrane functions including those that may control cellular differentiation.


A spectroscopic and electron microscopic examination of the highly condensed DNA structures formed by denaturation in Mg(ClO4)2. 1. Thermal denaturation in 1.5 M Mg(ClO4)2 of the  DNA from bacteriophage lambda results in four well-separated subtransitions, as monitored by the accompanying increase in absorbance. The midpoint of the hyperchromic spectrum is significantly lowered compared to either 1.5 M MgCl2 or 3.0 M NaClO4. 2. The first two subtransitions are associated with the melting of the A . T-richest regions of the lambda DNA, as revealed by electron micrographs following fixation with formaldehyde. 3. Commencing with the third subtransition, an unusual DNA structure is observed in electron micrographs. In this structure the A . T-rich half of the molecule appears completely condensed, whereas the G . C-rich half remains native. 4. During the fourth subtransition DNA molecules condense completely and eventually aggregate to form extremely high molecular weight particles containing centers of electron density. Tendrils of DNA, primarily duplex, radiate outward from these centers. 5. The aggregation may be reversed by the removal of magnesium. The intramolecular condensation may be at least partly reversed by increasing the Mg(ClO4)2 concentrations to saturating levels.


Poly(A) polymerase activity during cell cycle and erythropoietic differentiation in erythroleukemic mouse spleen cells. Poly(A) polymerase activity was studied in lysates of cultured murine erythroleukemic cells (Friend cells). Incorporation of ATP into acid-precipitable products is dependendent on the presence of Mn2+ or Mg2+ and of an RNA primer. The reaction is specific for ATP as the substrate (KM=290 290 micron, it is not inhibited by actinomycin D and only slightly interferred with by ethidium bromide. Cordycepin 5'-triphosphate and sodium pyrophosphate inhibit the enzyme activity. The chain length of the products of the reaction is dependent on the primer concentration and reaches up to 30 nucleotides. Poly(A) polymerase activity is low in resting (G1 phase) cells 75 nmol ATP incorporated/h per 10(6) cells) and increases to a level about twice as high in early S phase of the cell cycle. A possible model for regulation of enzyme activity is discussed. Polymerase activity in the early phase of erythropoietic differentiation of the cells induced by butyric acid does not show any difference in comparison to untreated controls. A decrease in enzyme activity to levels characteristic for cells in G1 phase accompanies shutdown of cell growth in the course of the ongoing differentiation. Analysis of the DNA content of the cells revealed that erythropoietic differentiation of Friend cells induced by butyric acid is characterized by arrest of the cells in G1 phase of the cell cycle. Poly(A) polymerase activity in erythroleukemic cells is thus controlled only by the phase of the cell cycle; it is not affected by changes in gene expression during erythroid differentiation.


Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro. 1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. The products of synthesis are short (Mr less than 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate/urea, guanidinium . HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase.


The effect of different buffers on terminal deoxynucleotidyl transferase activity. The polymerization of dATP, dCTP, and dGTP onto the defined length initiator, d(pA)10, has been carried out in four buffers. The relative effectiveness of the buffers for the polymerization of each deoxynucleoside triphosphate decreased in the order: cacodylate, 2(N-morpholino)ethane sulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and Tris. With the poorer buffers, activity could be increased by the addition of KCl: this effect is not primarily due to an increase in ionic strength. With dGTP as the substrate, but not with dATP or dCTP, activity increased when the concentration of the more active buffers was raised beyond 0.2 M.


Kinetic mechanism of the hydroxypyruvate-lactate dehydrogenase-NADH system. Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.


Purification of beef-heart cytochrome c oxidase by hydrophobic interaction chromatography on octyl-Sepharose CL-4B. 1. Hydrophobic interaction chromatography on Octyl-Sepharose CL-4B is used as a new and simple method for the preparation of large amounts of beef-heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). 2. The method involves only one cycle of (NH4)2SO4 fractionation before the material is applied to the column. After washing with 10% cholate and 1.5% Tween 80, elution of the enzyme is accomplished with 1% Triton X-100. 3. The enzyme so prepared contains about 10 nmol heme alpha/mg protein and about 0.2% phospholipid. 4. Characterization of the enzyme has been made with optical and EPR spectroscopy and polyacrylamide gel electrophoresis. The preparation appears by these criteria to be at least as good as other purified enzyme preparations. 5. The turnover rate at infinite cytochrome c concentration in 0.1 M sodium phosphate buffer and 0.5% Tween 80 at pH 6.1 is 80 s-1 per functional unit of the enzyme. A more than three-fold activation could be obtained by the addition of phosphatidylcholine at neutral pH.


Oscillatory kinetics of the peroxidase-oxidase reaction in an open system. Experimental and theoretical studies. 1. The oscillations in the peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7)-catalyzed reaction between NADH and O2 are undamped when the reaction is carried out in a system open to both substrates and when 2,4-dichlorophenol and methylene blue are present in the solution. 2. The waveform of the oscillations changes when the concentration of peroxidase is varied. 3. The waveforms obtained experimentally can be simulated by a branched chain reaction model in which the branching is quadratic. 4. A correlation between the present knowledge of the reaction and the model can be made by combining well established and hypothetical reaction steps into a few reaction schemes. A selection among schemes however, is not possible at the present time. 5. Compound III participates in the reaction as an active intermediate. This is possible because dichlorophenol stimulates the break down of compound III.


Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts. Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.


Dipeptidyl carboxypeptidase from human seminal plasma. Dipeptidyl carboxypeptidase (angiotensin I converting enzyme) was purified from human seminal plasma. The apparent relative molecular mass determined by gel filtration on Sephadex G-200 was 330 000. The pI in isoelectric focusing was 4.6--5.0 and the optimum pH 7.7--8.0. The enzyme is activated by chloride. These properties are similar to those reported for the lung enzyme. The specificity is that of a carboxypeptidase releasing dipeptides. A study of different substrates showed the activity to be highest with Z-Leu-Gly-Gly, followed by Z-Phe-His-Leu greater than bradykinin greater than Bz-Gly-Gly-Gly greater than Boc-Phe-Ala-Pro greater than Bz-Gly-His-Leu greater than angiotensin I.


Selective precipitation of 32Pi onto filter papers. Application to ATPase and cyclic AMP phosphodiesterase determination. A simple and selective method is described for the isolation of 32Pi based on the precipitation of the phosphomolybdate complex with triethylamine. Precipitation takes place on filter paper, which are then washed with a solution containing ammonium molybdate and triethylamine to remove other radioactive phosphates. Very large numbers of samples and very small sample volumes can be accommodated easily. The use of this method to measure ATPase and cyclic AMP phosphodiesterase activity is demonstrated.


Iron: a possible heterotropic effector of prolyl hydroxylase. The mechanism of ferrous ion binding to prolyl hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) was studied according to Koshland's curve-fitting procedure (Cornish-Bowden, A. and Koshland, Jr., D.E. (1970) Biochemistry 9, 3325--3336). The calculated data obtained by means of the unrestricted Adair equation were found to provide an adequate fit with experimentally obtained values, whereas those obtained on the basis of Michaelis-Menten kinetics did not. This suggests that prolyl hydroxylase could be an allosteric enzyme under positive heterotropic control.


Pilot scale purification of alpha-galactosidase A from Cohn fraction IV-1 of human plasma. Human plasma alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified 7000-fold over plasma levels from Cohn Fraction IV-1. The yield per kg starting material averaged 11 000 units (nmol galactose liberated per h) and the specific activity was about 600 units per mg protein with 4-methylumbelliferyl-alpha-D-galactoside. The ratio of 4-methylumbelliferyl-alpha-galactosidase to ceramide trihexosidase activities was 6.2. Both activities were heat labile and exhibited the same relative mobilities on polyacrylamide gel electrophoresis. Enzymatic activity was stable for at least 4 months at 4 and -20 degrees C. The endotoxin concentration of this preparation averaged 0.26 mg per mg protein.


Specific cleavage of reduced and S-carboxamidomethylated neurophysin II by the collagenase of Clostridium histolyticum. Purified collagenase of Clostridium histolyticum was shown to cleave reduced and S-carboxamidomethylated bovine neurophysin between Cys-13 and Gly-14. The scission resulted in formation of two separable fragments: a smaller peptide arising from residues 1 through 13, and a larger peptide comprising the remainder of the residues of the protein. By dansylation procedures, the smaller peptide was shown to have amino-terminal alanine as expected from the sequence of neurophysin II, and the larger peptide had amino-terminal glycine as anticipated. These results show that collagenase indeed cleaves bovine neurophysin II in accord with the specificity postulated for that enzyme, i.e., scission between -X-Gly- in a sequence of -Pro-X-Gly-Pro-Y-. This result, obtained with a non-collagenous protein substrate, is further confirmation of the specificity of collagenase as established by its action on collagens and on synthetic oligopeptides.


Isolation procedure and some properties of myeloperoxidase from human leucocytes. 1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.


Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase. Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.


Regulatory role of insulin in the degradation of low density lipoprotein by cultured human skin fibroblasts. The degradation of 125I-labeled low density lipoprotein by cultured human skin fibroblasts was enhanced 25% by preincubation of cells with insulin. This effect of insulin appeared to be mediated via stimulation of low density lipoprotein binding to its cell surface receptor, since binding and subsequent internalization of low density lipoprotein were stimulated to a similar extent as was degradation. In addition, insulin enhanced binding of low density lipoprotein at 4 degrees C, at which temperature internalization of the lipoprotein does not occur. A similar effect of insulin on the interaction of very low density lipoprotein with cultured fibroblasts was observed. Insulin-induced changes in the degradation of low density lipoprotein and very low density lipoprotein appeared to be a function of the change in lipoprotein binding. Thus, insulin may play a role in the regulation of low density lipoprotein and very low density lipoprotein degradation by peripheral cells by influencing the receptor-mediated transport of these lipoproteins.


Surface exposure of apolipoproteins in high density lipoproteins. I. Reactivities with agarose-immobilized proteases. The exposure of apolipoproteins at the surface of human plasma high density lipoproteins (HDL) was assessed by their accessibility to agarose-immobilized forms of trypsin and chymotrypsin. Proteolysis of lipid-free apolipoproteins and the lipoprotein subfractions HDL2 (d = 1.08--1.125 g/ml) and HDL3 (d = 1.125--1.195 g/ml) that differ in lipid-to-protein ratio was compared by polyacrylamide gel electrophoresis and isoelectric focusing of the apolipoproteins and peptide fragments and by quantitation of the various carboxyl-terminal groups formed. Gel filtration of the proteolyzed lipoproteins on Sephadex G-150 column indicated that more than 90% of the apolipoproteins and peptides remain associated with lipoprotein complexes. Proteolysis of lipoproteins occurred more slowly and with less fragmentation of the lipoproteins and apolipoproteins than proteolysis of thelipid-free apolipoproteins or the proteolysis of lipoproteins by soluble proteases reported by other investigators. The difference in lipid content of HDL2 and HDL3 made little difference in their proteolysis. Proteolysis of the lipoproteins by agarose-trypsin was more rapid at 37 degrees C than at 22 degrees C, but the proteolytic products were similar and differed from the products from the lipid free proteins. Peptide fragments from lipoproteins were larger than those from lipid-free proteins, which suggests masking of potentially cleavable groups by lipid. The amounts (mol/g protein) of new carboxyl-terminal tyrosine and phenylalanine released by agarose -chymotrypsin were much greater from the lipid-free proteins, but about 3/4 of the tryptophan residues were inacessible in both lipoproteins and lipid-free proteins. In agarose-trypsin digestion, lysine residues were slightly more masked than arginine in the absence of lipids and much more so in the lipoproteins. However, in the lipoproteins apoA-II, which contains lysine but no arginine, was cleaved more rapidly and extensively by agarose-trypsin than apoA-I.


The degradation of very low density lipoprotein by the extrahepatic tissues of the rat. The supradiaphragmatic rat was used to investigate the metabolism by the extrahepatic tissues of endogenous plasma VLDL of d less than 1.006 g/ml. The demonstration that, at 20, 30 and 40 min after the isolation of the supradiaphragmatic rat, the VLDL lose respectively 29, 54, and 63% of their triglyceride provides evidence for the suitability of this preparation for the investigation of VLDL degradation. At all time intervals after the isolation of the supradiaphragmatic rat, VLDL triglyceride loss was accompanied by similar losses of cholesterol, protein and phospholipid, with the result that the percentage by weight composition of the residual VLDL remained unaltered. By subfractionation of the VLDL, a group of particles with an Sf range of 20--60 were isolated that, when compared with total VLDL, were enriched in their cholesterol (P less than 0.02), protein (P less than 0.001) and phospholipid (P less than 0.01) content. However, these particles represented only a small percentage of the total VLDL mass. Furthermore, their amount was not increased in the circulation of the supradiaphragmatic rat. The amount of IDL (d = 1.006--1.019 g/m) and of LDL (d = 1.019--1.063 g/ml) was increased in the supradiaphragmatic rat and a part of the total cholesterol and protein lost from the VLDL could be accounted for by the increases in these constituents in the IDL and LDL fractions. It is suggested that, although the liver probably takes up partial degradation products of VLDL in the intact animal, the extrahepatic tissues alone can metabolize VLDL to LDL of d = 1.019--1.063 g/ml. The lipoprotein particles taken up by the liver in the intact animal appear most likely to be those of Sf greater than 100.


Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteins. The apoprotein of human serum low density lipoproteins was reduced and carboxymethylated and then cleaved by cyanogen bromide (CNBr). The peptides which were produced from this cleavage (90% yield, based upon loss of methionine) were resolved by SDS polyacrylamide gel electrophoresis into 10 major bands, each having an amino acid composition very similar to that of intact reduced and carboxymethylated LDL apoprotein. The fractionation of the CNBr fragments by preparative gel filtration was dependent upon the nature of the eluting solvent. NH4OH and SDS solvents eluted all of the material in the void volume. In 6 M guanidinium chloride solvents several peaks were, however, resolved, each having an amino acid composition similar to that of the unfractionated products. Whereas no NH2-terminal was detected in reduced and carboxylmethylated LDL apoprotein, automated Edman degradation of the protein following treatment with CNBr revealed the presence of several NH2-termini. The results suggest that LDL apoprotein may be made of segments of, at least, very similar amino acid composition and that both the protein itself and derivative fragments have a great tendency to aggregate even in denaturing solvents.


Very low density lipoprotein stimulation of triglyceride accumulation in rat preadipocyte cultures. Exposure of cultured rat epididymal preadipocytes to human very low density lipoproteins (VLDL) resulted in the rapid accumulation of large amounts of cellular triglyceride which was accompanied by the appearance of numerous large cellular lipid inclusions. Addition of heparin produced a two-fold stimulation of lipoprotein induced triglyceride accumulation. Supplementation of the growth medium with either low density lipoprotein, oleic acid or artificial triglyceride emulsion did not produce cellular triglyceride levels equivalent to that obtained with VLDL. Fibroblastic cells from rat skin and lung did not accumulate triglycerides when exposed to VLDL and heparin.


Affinity electrophoresis of proteins interacting with Blue dextran. Interaction of several enzymes (pyruvate kinase, myokinase, creatine kinase, aldolase, malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase) and other proteins (bovine serum albumin and ovalbumin) with Blue Dextran was studied by means of affinity electrophoresis in polyacrylamide gels. A decrease of electrophoretic mobility of enzymes in affinity gels was dependent on Blue Dextran concentration and in some cases, dissociation constants of the protein-immobilized dye complexes could be calculated. Affinity electrophoresis in the presence of Blue Dextran reveals in some cases additional bands of isoenzymes, as compared with the control gels (without Blue Dextran).


The state of copper in Neurospora laccase. 1. Neurospora crassa laccase has been prepared from the growth medium and studied by optical absorption, circular dichroism and electron paramagnetic resonance (EPR) spectroscopy. The molecular weight, the copper content and the amino acid composition have also been determined. 2. The molecular weight as determined by gel filtration in 6 M guanidine hydrochloride and by sodium dodecyl sulfate gel electrophoresis is found to be 64 000. The enzyme contains 3.8 copper ions per 64 000. 3. The visible and the near ultraviolet difference absorption spectrum shows two maxima, at 330 and 595 nm, and a shoulder at about 720 nm. The circular dichroism spectrum between 300 and 760 nm contains five bands in the oxidized enzyme. After reduction of the enzyme with ascorbate there remains only a band at 305 nm. 4. EPR measurements show that 52% of the total copper in the protein is paramagnetic. Two EPR signals of equal intensity with different hyperfine splitting constants, of 9 and 18.5 mT, are present, which are assigned to Type 1 Cu2+ and Type 2 Cu2+, respectively, as found in other blue copper-containing oxidases.


Specificity of the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase and to a series of other calcium-binding proteins. Trifluoperazine inhibits the activation of phosphodiesterase by binding to the calcium-dependent activator. To determine further the specificity by which trifluoperazine binds to activator, we compared the binding of trifluoperazine to activator prepared from several species and tissues and to a number of other calcium-binding proteins devoid of activator activity. Trifluoperazine binds to activator prepared from human, bovine, rat and rabbit brain and from chick embryo fibroblasts. In each case, the binding of trifluoperazine to activator was qualitatively similar and related quantitatively to the ability of the preparation to activate phosphodiesterase. Of the other calcium-binding proteins examined, namely, troponin-C, S-100 protein, phospholipase A, phospholipase B and myosin light chain, only troponin-C displayed any significant calcium-specific binding of trifluoperazine. The binding to troponin-C, however, appeared to be different from the binding to activator; whereas the binding of trifluoperazine to actovator showed no cooperativity, the binding to troponin-C showed positive cooperatively. These results and earlier data showing that trifluoperazine fails to bind to a variety of other proteins, indicate that the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase is selective and suggest that this binding may explain some of the biochemical and pharmacological actions of this antipsychotic agent.


Secretion of epidermal growth factor. The role of calcium in stimulcule during secretion. Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca2+ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of alpha-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth facto; occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.


Hormone action at the membrane level. VII. Stimulation of dihydroalprenolol binding to beta-adrenergic receptors in isolated rat heart ventricle slices by triiodothyronine and thyroxine. Thyroxine (T4) and triiodothyronine (T3) increase the number of beta-adrenergic receptors in heart ventricle slices. A short term effect reaches a maximum by 1.5-2 h and requires added amino acids for consistent results. The apparent Km for the L-T3 effect is 15 pM. This effect, measured by an increase in stereospecific binding of (-)-[3H]dihydroalprenolol is not inhibited by cycloheximide or puromycin and is produced more effectively by L-T3 than D-T3. However, cycloheximide nearly completely inhibits protein synthesis in the 2-3 h incubation time. T3 also gives a small inhibition of protein synthesis during this time interval. The early effect of T3 stimulation of dihydroalprenolol binding is considered to be a post-translational event shereby T3 enhances the transport of existing beta-adrenergic receptors from the cytosol into the membrane. A long term (15 h) stimulation of dihydroalprenolol binding to ventricle membranes is also produced by L-T3. This effect is stereospecific, is inhibited by cycloheximide, and is believed to be a transcriptional-translational event leading to the synthesis of new beta-adrenergic receptors by T3.


Dissociation of lutropin-induced loss of testicular lutropin receptors and lutropin-induced desensitization of testosterone synthesis. The relationship between changes in testicular lutropin receptors, as measured by specific binding of 125I-labeled human chorionic gonadotropin, and testosterone synthesis in response to lutropin (testicular responsiveness) was studied in intact and hypophysectomized rats. Administration of a single 200-microgram dose of ovine lutropin to intact rats results at 3 days in a 58% decrease in lutropin receptors associated with a parallel decrease in testicular responsiveness. A single 30-microgram dose of lutropin to intact rats resulted in a comparable decrease in lutropin receptors with a transient increase in testicular responsiveness. Rats receiving twice-daily injections of 15 microgram lutropin for 10 days exhibited a 48% decrease in lutropin receptors by day 3 which persisted during the 10-day treatment period, but was accompanied by a progressive increase in testicular responsiveness to lutropin. Hypophysectomy resulted in an 80% loss of receptors and a 72% loss in responsiveness 7 days after surgery. Daily treatment with lutropin initiated immediately following surgery resulted in a further dose-dependent decrease in lutropin receptors and a dose-dependent increase in testicular responsiveness. Loss of lutropin receptors was not due to occupancy of the receptor by exogenous lutropin. These studies demonstrate a dissociation between the negative regulation of lutropin receptors and testicular responsiveness to lutropin. Furthermore, the studies in hypophysectomized rats indicate that lutropin is the only hormone essential for maintenance of steroidogenesis and that this is independent of lutropin receptor concentration.


Mercury complexes of thionicotinamide adenine dinucleotide. One-to-one mercury complexes of thionicotinamide adenine dinucleotide (TNAD+) were prepared by using HgSO4 and Hg(CH3 COO-)2. Optical absorption spectroscopy indicated that the mercury probably binds to the TNAD+ through the thio-keto group on the pyridine ring. X-ray diffraction patterns of crystals of mitochondrial malate dehydrogenase soaked in solution containing TNAD+ . mercury complex indicated binding and the X-ray intensity differences are different from mercurials alone.


[Kinetic characteristics of thiamine diphosphate biosynthesis by thiamine pyrophosphokinase from rat liver]. The kinetic analysis of bisubstrate enzymatic reaction catalysed by electrophoretically homogenous thiamine pyrophosphokinase (EC 2.7.6.2), isolated from rat liver has been carried out. Kinetic studies of the initial rates in the absence of the products and inhibition by the reaction products as well as the data from the equilibrium dialysis suggest that the reaction proceeds through the formation of a ternary enzyme-substrate complex. The combination with substrates and release of the products appears to be highly ordered. A possible scheme of the reaction mechanism is discussed.


[Protein inhibitor of the retinal cyclic nucleotide phosphodiesterase: its localization in the outer segment of a photoreceptor]. The content of a protein inhibitor of the cyclic nucleotides phosphodiesterase (PDE) in different retinal preparations as well as its distribution in the subfractions of rod outer segments (ROS) was studied. The content of protein inhibitor of PDE in different preparations of the retina was found to correlate with the rhodopsin content. The distribution of this protein over different ROS subfractions appeared to be exactly the same as that of rhodopsin, the content of protein inhibitor of PDE being more than a half of its content in the native ROS. The protein inhibitor of PDE could be easily washed out from the ROS fractions. It is concluded that the cattle protein inhibitor of PDE is localized in ROS, and is absent in the other retinal layers.


[Protein phosphorylation in normal and neoplastic hepatocytes]. Partially purified preparations of protein kinase were isolated from the cytoplasm and nuclei of rat liver and hepatoma 27 and characterized in terms of their substrate specificity. The protein kinases from normal liver and hepatoma revealed some differences in phosphorylating protein substrates. Hepatoma protein kinases were found to phosphorylate arginine-rich histones (H3, H4); differences in phosphorylation of histone H1 were revealed. Hepatoma protein kinases phosphorylated the C-terminal fragment of histone H1, whereas normal liver protein kinases produced no such effect. It was assumed that the phosphorylation of histone H1 in rapidly dividing and resting cells is operated through different channels.


[Study of insulin receptors on liver and hepatoma cell plasma membranes]. Plasma membranes (PM) of helathy rats as well as those hepatomas and PM of tumor-bearing rats were found (radioreceptor assay) to have apparently two groups of receptors for insulin with a high and low affinity for the hormone respectively and different insulin-binding capacities. Kass values of the receptors with a high affinity for insulin are drastically decreased in the PM of ascites Zajdela hepatoma (AZH) and of solid hepatoma 27 (SH-27) as well as in the liver of SH-27-bearing animals, but not in the liver of the AZH-bearing rats. Kass values of the receptors with a low affinity for insulin in the PM of AZH and in those of the liver of AZH-bearing rats appear nearly normal. In contrary, above Kass the receptors in the PM of SH-27 and SH-27-bearing animals are significantly decreased. The insulin-binding capacity of the receptors with a high affinity for the hormone in the PM of SH-27, AZH and the liver of both tumor-bearing rats is shown to be significantly higher than that in the PM of normal animals. The same property of the receptors with a low is affinity in the PM of the hepatomas and theliver of their hosts is also increased, especially in the PM of SH-27 and of the liver of SH-27-carring rats.


[Redox potentials of some metalloproteins]. The standard redox potentials of soluble cytochromes c isolated from the green alga Chlorella and the blue-green algae Spirulina and Aphanezomenon were determined by potentiometric titration and found to be equal to +380 mB, +330 mB and +357 AB, respectively. The standard redox potentials of plastocyanin preparations from Pisum sativum and Atriplex leaves were also determined and found close to those of soluble cytochromes c, i. e. +395 mB and +375 mB, respectively. The metalloproteins studied were shown to belong to monoelectron carriers operating at the donor sites of photosystem I.


[Comparative study of B890 pigment-lipoprotein complexes from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple photosynthesizing bacteria]. Pigment-lipoprotein B890 complexes containing reaction center and "light-focusing" bacteriochlorophyll a were isolated from photosynthetic membranes of sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria after the treatment with Triton X-100. The molecular weights of complexes were evaluated using several methods (200 000-300 000). By means of electron microscopy the sizes of complexes were found to be about 80 A. On the air-water interface hexagonal packing of complexes was observed. The chemical compositions of complexes are very similar except bacteriochlorophyll a whose specific content is somewhat higher in Chromatium minutissimum. The protein composition of complexes was studied and the molecular weights of proteins were estimated by SDS-gel electrophoresis. The results obtained show significant similarities in molecular organization of B890 complexes isolated from sulfur (Chromatium minutissimum) and non-sulfur (Rhodopseudomonas palustris) purple bacteria.


[Effect of growth conditions on the activity of the enzymes of cyclic 3':5'-AMP synthesis and decay in phototrophic bacteria]. Activity, ratio and summary content of cyclic AMP enzymes, adenylate cyclase and phosphodiesterase varied depending on growth conditions of phototrophic bacteria (Rhodospirillum rubrum and Rhodopseudomonas palustris). It suggests, that membrane-bound and soluble enzymes carry different functions. The increase of adenylate cyclase under chaning growth conditions was usually accompanied by the increase of phosphodiesterase. Sharp increase of both enzymes activity was observed when bacteria were growth in aerobic conditions. The activity of both enzymes in chromatophores was 2.8-fold higher when bacteria were grown in the light in anaerobic conditions, than in chromatophores of bacteria grown under stationary aerobic conditions in the light. It is suggested that 3':5' AMP can participate in autotrophic carbon assimilation or in the synthesis of pigments and other components of bacterial photosynthetizing apparatus. Substitution of NH4+ into NO3- and glutamate under the growing of R. rubrum in anaerobic conditions in the light resulted in the increase of the enzymes activities, which is the evidence of possible role of 3':5' AMP in mineral nitrogen uptake and nitrogen fixation. Glutamate concentration of 4 g/l stimulated the enzymes both in vivo and in vitro. The data obtained suggest that 3':5' AMP can carry multiple functions, participating in regulation of a number of metabolic processes in photorophic bacteria.


Urinary content of 3',5'-adenosine monophosphate (cAMP) and creatinine in full-term and premature infants. Cyclic 3',5'-monophosphate (c-AMP) and creatinine were measured in full-term and premature infants. In normals, values for c-AMP were highest on days 1 and 2 (1.3-1.5 nmol/ml) and decreased thereafter. In prematures the c-AMP values were significantly lower than in the full-term infants. There was a rough correlation with birth weight. Creatinine values were closely reflected by the c-AMP levels. Changes found in neonatal jaundice, hypocalcaemia, respiratory distress syndrome, and anoxia could be attributed to prematurity. No diurnal variation in c-AMP values was found.


Calcium and 25-hydroxyvitamin D3 binding proteins in human placenta. The placental translocation of calcium is a well-known process, but the underlying mechanisms remain unknown. Placental cytoplasmic proteins capable of binding calcium and the active metabolites of vitamin D3 were sought in these studies. Cytosol from full-term human placenta was prepared by ultracentrifugation of homogenates and tested for binding. Partial purification by column chromatography revealed the presence of two discrete proteins which bound specifically 45Ca and 3H-25-hydroxyvitamin D3 and having approximate molecular weights of 12,000 and 67,000, respectively. These placental proteins may be intermediaries in the process of placental calcium translocation.


[Change in the concentration of nucleic acids and cAMP in neurogenic dystrophies of the stomach]. There proved to be a reduction of the RNA content in the tissue of the rat stomach in its dystrophic affection induced by traumatization of the duodenum, this pointing to the reconstruction of the cell genetic apparatus under conditions of extreme stimulation. Under the same experimental conditions there was a pronounced decrease of the cAMP level in the gastric mucosa of rabbits. The significance of the changes in the mechanism of the development of destructive and metabolic disturbances in the neurogenic dystrophic affections induced by extreme stimulation is discussed.


[Effect of heparin on the activity of liver microsome oxidative enzyme systems in healthy rats and rats with experimental glomerulonephritis]. In experiments on mongrel rats it was found that heparin in doses of 2, 5, and 10 mg/kg had an inducing effect on the oxidative systems of hepatic microsomes: hexenal test period was shortened, cytochrome P450 content increased, relative liver weight rose; the activity of histochemically-detectable NAD-enzymes of hepatocytes became greater. Heparin was capable of considerable (2-3-fold) stimulation of antitoxic activity of the liver reduced in experimental glomerulonephritis. The effect of heparin on the antitoxic function of the liver did not correlate with its effect, on the blood coagulation system.


[Activity of gluconeogenetic enzymes of rat kidney cortex during acute hypoxia]. The activities of gluconeogenic enzymes of the rat kidney cortex was studied after exposure to lowered atmospheric pressure (200 mm Hg) for 3 hours. The hypoxic stress was found to cause an increase in the activities of phosphoenolpyruvate carboxykinase and alanine aminotransferase, but failed to affect significantly the activities of fructose-1,6-diphosphatase, glucose-6-phosphatase, and aspartate aminotranspherase. The ratio of glucose-6-phosphatase/hexokinase activities was increased under these conditions.


[Regulatory role of DOPA and components of the cyclic adenosine-3',5'-monophosphate system]. It was shown on albino mice that when DOPA-3H (20 muCi/mouse) was administered before nonradioactive DOPA (1 mg/mouse) tritium accumulation in the tissue of Harding-Passi's melanoma of these mice proved to increase. Melanoma radioactivity in this experimental group was double that in the tumour tissue of the animals to which DOPA-3H alone was administered. Examination of the adenylate cyclase, phosphodiesterase activity and of the level of cAMP in melanoma of mice 2 hours after DOPA administration (1 mg/mouse) showed accumulation of cAMP and an increase in the phosphodiesterase activity; as to adenylate cyclase activity--it fell. It is suggested that DOPA realizes its effect not only as melanin precursor, but also through the cAMP system, influencing the melanogenesis enzymes activity.


[Experimental influenza caused by an incomplete form of the agent]. Incomplete form of the influenza virus obtained in accordance with Nayak's method was administered intranasally to mice CBA and C57BL. From the lung tissue of the infected mice the causative agent could be isolated for 45 days, and from the other internal organs--the first hours after the infection only. In morphological investigation of the lungs of animals infected with an incomplete form of the influenza virus a prevalence of the proliferative component against the background of inflammatory changes was noted. Three months after the infection limited lymphoid formations consisting of monomorphic cells with hyperchromic nuclei were defined in the lung tissue. Marked proliferation of the alveolar and bronchial epithelium was observed later; considerable anaplasia of the cells was noted in the papillomatous structure of the alveolar and bronchial epithelium. Glomangioma of the mesentery was observed among affections of other internal organs in 18.7% of mice CBA.


[Electron microscopic demonstration of adenylate cyclase in rabbit small intestine enterocytes doubly stimulated by cholera toxin and sodium fluoride]. Cytochemical investigations showed adenylate cyclase in the rabbit small intestine enterocytes to be activated both with cholera toxin and sodium fluoride. Following double stimulation of adenylate cyclase in the intestinal enterocytes by the mentioned two substances maximal critical levels of cAMP were attained resulting in self-inhibition of adenylate cyclase; in this case only a low adenylate cyclase activity, if any, could be demonstrated by electron microscopy.


[Leukocyte migration inhibition reaction in trophoblastic tumor patients]. Inhibition of leukocyte migration was studied in 40 patients with trophoblastic tumours under the effect of protein extract from chorionepithelioma. A marked inhibition of leukocyte migration was revealed in patients with signs of active tumour process before and during the treatment. Four patients in whom no inhibition was revealed either before or during the treatment were an exception. In the majority of the patients the inhibition effect was absent against the background of clinical well-being achieved as a result of surgical and chemotherapeutic, or chemotherapeutic treatment alone. Leukocytes of healthy donors failed to respond to the tumour extract in all 24 cases.


Long-term cultivation of plasma cell leukemia cells and autologous lymphoblasts (LCL) in vitro: a comparative study. Two long-term cell lines were established in vitro from the peripheral blood of a patient with plasma cell leukemia: one line with plasma cell proliferation, the other with lymphoblastoid cell proliferation (LCL). The 9-month-old plasma cell line showed the typical morphology of plasmoblasts. The cells neither had B- nor T-lymphocyte characterisitics, were EBV negative, and showed aneuploidy with various marker chromosomes, including the 14 q+ marker. The cytogenetic findings indicate a monoclonal proliferation of the plasmacells. No tumor growth in thymusless nude mice could be induced upon intracranial inoculation with these cells. In contrast, the autologous LCL, cultured after addition of exogenous EBV, showed the characteristic markers of lymphoblastoid cells, with the typical morphology of pear- and handmirror-shaped lymphoblasts, growing in clumps. They had C3- and Fc-receptors, surface-Ig, E-rosette-negativity, a diploid karyotype, and EBV dependent macromolecule synthesis. They lymphoblastoid cells produced intracranial tumors in nude mice in 8 out of 8 attempts.


Specific immunity to human papilloma virus (HPV) in patients with genital warts. A study of human papilloma virus (HPV) specific cellular and humoral immunity in 30 patients with genital warts is reported. By in vivo testing with purified, inactivated plantar wart virus, a cell-mediated immunity to HPV was determined in 60% of patients. Circulating antibodies, evaluated by immunofluorescence testing, were rare, but these increased after an intradermal test had been carried out, especially in patients with a positive skin test, suggesting a booster effect. No significant difference was found between this group of patients and those having skin warts. Our results showed a specific immune response to HPV in most patients, confirming the role of the viral agent in the induction of genital warts.


Strychinine binding associated with synaptic glycine receptors in rat spinal cord membranes: ionic influences. Ammonium salts of some anions decrease the potency of glycine in inhibiting (3H)strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC50 of glycine in inhibiting the (3H) strychnine binding, their capacity to reduce the (3H) strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of (3H)strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting (3H)strychnine is not. Binding of (3H)strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li+, K+, and Na+ up to 150mM decrease (3H)strychnine binding, while higher concentrations of the cations increase (3H)strychnine binding. Inhibition by glycine of (3H)strychnine binding is enhanced by low concentrations of these cations.


An ultrastructural study into the effects of pentobarbitone on synaptic organization. A series of adult male rats was analyzed to test the effects of varying doses of pentobarbitone sodium on the ultrastructure of synapses in the molecular layer of the parietal cortex. The rats were divided into the following categories: unanaesthetized stunned, unanaesthetized cannulated and those subjected to 40, 80, 160, 300, or 400 mg/kg pentobarbitone. All material was examined both qualitatively and quantitatively after aldehyde-OsO4 or ethanolic phosphotungstic acid (E-PTA) treatment. The principal qualitative observations were: the preponderance in the unanaesthetized stunned and 160-400 mg/kg material of a variety of intraterminal profiles including synaptic vesicles, mitochondria, coated vesicles, tubular profiles, vacuoles, cisterns and double membrane profiles; the presence of exocytotic sites along the presynaptic membrane in the unanaesthetized stunned and cannulated material; the presence of endocytotic sites over the limiting membrane of the terminal away from the cleft in the unanaesthetized stunned and 160-400 mg/kg material; the prominence of the presynaptic network in the unanaesthetized and 40 mg/kg E-PTA material; discontinuity of the cleft material in the 40-160 mg/kg material. Findings to emerge from the quantitative aspect of the study show that pentobarbitone influences synaptic curvature, with a marked increase in curvature negativity over the 0-80 mg/kg dose range and a decrease in negativity at higher dose levels. The increase in curvature negativity is accompanied by an increase in synaptic length and dense projection numbers, with a consonant increase in the perimeter and area of the presynaptic terminal. Reversal of the negativity trend at higher dose levels is parallelled  by reversal of these accompanying trends. Both sets of findings can be accounted for by membrane recycling within the terminal, supporting a membrane redistribution hypothesis.


Muscimol binding in rat brain: association with synaptic GABA receptors. [3H]Muscimol binding to crude synaptic membrane fractions of the rat central nervous is saturable with a high affinity dissociation constant of 2.2 nM. The regional distribution of this binding in rat brain and the effects of freezing, sodium and Triton X-100 are similar to those previously reported for [3H]GABA binding to the the synaptic GABA receptor site. Also, the substrate specificity of [3H]muscimol binding is identifical to that observed for the GABA receptor. Thus, [3H]muscimol is displaced, stereospecifically, only by those drugs and amino acids which are known to neurophysiologically interact with the synaptic GABA receptor but is unaffected by agents which activate or inhibit other neurotransmitter receptors. This suggests that the behavioral effects observed after the systemic administration of muscimol are probably the result of GABA receptor activation.


Small mode miniature end plate potentials are increased and evoked in fatigued preparations and in high Mg2+ saline. Amplitude histograms of miniature end-plate potentials (MEPPs) from small sartorius fibers (6-20 micron diam.) of very small frogs were found to have multiple peaks. All MEPPs had the same time characteristics which demonstrates that they arose at the same junction. The amplitude of the smallest mode (s-MEPPs) was one-seventh that of the major mode (m-MEPPs), and in the unstressed preparation s-MEPPs composed only a few per cent of the total. After repetitive nerve stimulation (1 min at 20 Hz) the percentage of s-MEPPs was initially increased. Successive periods of stimulation gradually reduced the end-plate potential (EPP) (above 90% failures) to the same size as s-MEPPs. A 12.5 MM Mg2+ and half normal Ca2+ saline was also found to increase the percentage of s-MEPPs and reduce EPPs so that profiles of EPP amplitude histograms showed a class of small EPPs the same size as s-MEPPs. The data demonstrate that quanta the size of s-MEPPs were released with nerve stimulation in both the Mg2+ blocked and fatigued preparation.


Interneurons of sympathetic ganglia: divergent cyclic AMP responses and morphology in cat and cow. The biochemical properties of the small, intensely fluorescent (SIF) cells of bovine and feline superior cervical ganglia were determined by studying the cyclic AMP responses to incubation in vitro with adrenergic agonists: the morphological properties were studied by fluorescence histochemistry and electron microscopy. Incubation with 50 micron dopamine elicited cyclic AMP levels 524% of control values in the bovine ganglion, but only 152% in the feline ganglion (a value which is not statistically significant). Incubation with 50 micron isoproterenol produced an increase of 356% of control values in the cow, but no increase in the cat. SIF cells were classified into two types by fluorescence histochemistry. The bovine ganglion contained 23.7% Type I (solitary) SIF cells, and 76.3% Type II (clustered) cells, whereas the feline ganglion contained 99.5% Type II cells. Thus the feline ganglion lacks both Type I SIF cells and a dopamine receptor-adenylate cyclase complex. On the basis of biochemical and morphological evidence, we hypothesize that the Type I SIF cell is an interneuron, and infer that the lack of a response to dopamine stimulation in the feline ganglion is caused by a scarcity of interneurons in this species.


Angiotensin receptive neurones in the subfornical organ. Structure-activity relations. A microiontophoretic study was performed of the actions of angiotensin II and angiotensin fragments on neurones of the subfornical organ (SFO). Adult cats were anaesthetized and the SFO exposed for penetration by a multibarrelled micropipette. We found that angiotensin II-[2--8]-heptapeptide shows a significantly higher stimulation of firing rate compared to angiotensin II. Angiotensin II-[5--8]-tetrapeptide still produced an excitatory action on a single units. Both the action of the heptapeptide and the tetrapeptide were blocked by [sar1, Ala8]-angiotensin II (P 113). In contrast, angiotensin II-[6--8]-tripeptide failed to enhance the firing rate of the same neurones. Our data indicate that angiotensin II and some shorter chain peptide fragments can directly affect neurones of the SFO. The study may give new insight in structure-activity relations for angiotensin II. The results support the hypothesis that the subfornical organ is a receptor site which is available to this peptide.


Norepinephrine attenuation of amnesia produced by diethyldithiocarbamate. Rats given 680 mg/kh diethyldithiocarbamate, approximately one half hour before training in an inhibitory avoidance task, had impaired retention performance when tested one week after training. Intracerebroventricular or subcutaneous injections of norepinephrine administered shortly after training attenuated the disruptive effects of DDC on retention performance. The effect depended upon the footshock intensity used during training. NE(0.01 microgram) administered centrally attenuated the DDC induced retention deficit when animals were trained with a high (2 mA) but not a low footshock (0.5 mA). The effect of peripherally administered NE also varied with intensity of footshock. The lowest dose of subcutaneously administered NE (5 microgram/kg) was effective in attenuating DDC induced retention deficits only when animals were trained with higher footshock. Higher doses of NE (50 microgram/kg, 500 microgram/kg) were more effective when animals were trained with lower footshock.


[Influence of conditions of the acclimatization to cold on the thermogenesis without shivering in rats]. As indicated by a theophylline administration previously to a norepinephrine infusion, the nonshivering thermogenesis does not seem to be mediated by the cyclic AMP system in constant cold acclimated rats, in opposite to rats acclimated to discontinuous cold. However, in that last thermal condition, the cyclic AMP mediation was not observed in the brown adipose tissue and in the liver.


The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein. The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.


Efficacy of laboratory tests for the detection of enterotoxigenic Clostridium perfringens. Nineteen Clostridium perfringens strains with positive erythemal and ligated intestinal loop reactions, and 22 strains with negative reactions, originating from food-poisoning cases, were tested comparatively using the fluorescent antibody (FA), reversed passive hemagglutination (RPHA), and immunodiffusion (ID) tests. All the biologically positive strains were detected by the three immunological tests used. The FA test detected five additional strains among the biologically negative group which did not react in RPHA or ID tests. Sporulating culture supernatant fluids, after 13 to 17h of growth, were satisfactory for testing for the presence of enterotoxin by the RPHA and ID tests. The FA test was used on cell smears.


The lipoprotein lipase system: new understandings. Hypertriglyceridemia, a risk factor for premature atherosclerosis, may result from decreased use of plasma triglycerides by tissues. The removal of triglycerides is mediated by the enzyme lipoprotein lipase (LPL). Heparin releases LPL from tissues and post-heparin plasma lipolytic activity (PHLA) has been extensively used to elucidate  the mechanism of hypertriglyceridemia in various diseases. There is evidence to show that postheparin plasma contains enzymes other than LPL. Hence data on total PHLA are difficult to interpret. Availability of assays for the LPL component of PHLA has clarified equivocal findings in certain hypertriglyceridemic states. However, the LPL component is also heterogeneous. The LPL "isoenzymes" from various extrahepatic tissues behave differently under various metabolic conditions. Therefore, to understand properly the LPL system it is necessary to study the specific tissue LPL. Furthermore, the serum activator for LPL is now characterized. Its importance is evidenced by the recent discovery of a hypertriglyceridemic patient deficient in this apoprotein.


The effectiveness of prophylactic brain irradiation in small cell carcinoma of the lung: a Southwest Oncology Group study. Brain involvement in small cell carcinoma of the lung is a common phenomenon occurring in from 29 to 45% of patients. Because of this, it was suggested that prophylactic brain irradiation be made a part of treatment plans for small cell carcinoma. In December 1974, the Southwest Oncology Group (SWOG) began treating patients with combination chemotherapy and irradiation of both the primary lesion and whole brain. In two years, there were 390 patients entered into the study. In patients with extensive disease only 6 of 152 prophylactically irradiated patients developed CNS signs or symptoms of CNS recurrence. In limited disease, 6 of 88 prophylactically treated patients had CNS recurrence and in only 4 was this the site of initial failure. We feel prophylactic brain irradiation in small cell carcinoma of the lung is of benefit.


Carcinomatous versus radiation-induced brachial plexus neuropathy in breast cancer. A retrospective study was performed of 18 women in whom ipsilateral brachial plexus neuropathy developed after treatment for carcinoma of the breast. In the absence of metastatic tumor elsewhere, the only distinguishing feature between carcinomatous neuropathy and radiation-induced neuropathy was the symptom-free interval after mastectomy and radiation therapy. Women with an interval of less than a year have radiation-induced neuropathy. Brachial plexus exploration in difficult diagnostic situations will permit early treatment and avoid debilitating loss of function. Brachial plexus exploration for biopsy is safe and free of complications if performed carefully. Treatment of carcinomatous neuropathy is most likely to succeed if the tumor is hormonally sensitive, but radiotherapy may also be effective. Treatment of radiation-induced neuropathy remains largely ineffective.


Malignant fibrous histiocytoma: an analysis of 200 cases. The clinicopathologic findings in 200 cases of malignant fibrous histiocytoma (MFH) with follow-up information are presented. This tumor occurred principally as a mass on an extremity (lower extremity 49%, upper extremity 19%) or in the abdominal cavity or retroperitoneum (16%) of adults (peak incidence 61-70 years of age). It typically involved deep fascia (19%) or skeletal muscle (59%) and only rarely was confined to the subcutis without fascial involvement (7%). The MFH had variable morphologic features and frequently showed transitions from areas having a highly ordered storiform pattern to less differentiated areas having a pleomorphic appearance. The rate of local recurrence of the tumor was 44%, and of metastasis, 42%. Metastasis was most frequently to the lung (82%) and lymph nodes (32%). Factors that influenced the rate of metastasis included depth, size, and inflammatory component of the tumor. Tumors that were small, superficially located, or had a prominent inflammatory component metastasized less frequently than larger, more deeply located tumors. In our experience the MFH is the most common soft tissue sarcoma of late adult life, and many tumors previously diagnosed as pleomorphic variants of liposarcoma, fibrosarcoma, or rhabdomyosarcoma are probably examples of MFH. Although the histogenesis of this neoplasm remains controversial, we feel it is best regarded as a primitive and pleomorphic sarcoma showing partial fibroblastic and histiocytic differentiation, as reflected by collagen production and occasional phagocytosis.


Mediastinal paragangliomas (aortic body tumor): a report of four cases and a review of the literature. Four cases of paraganglioma originating in the supra-aortic or aortico-pulmonary bodies are described. A review of the reported examples of aortic body tumors and a study of these four cases indicate that there is a high incidence of aggressive tumor growth in the mediastinum, with resultant important morbidity or death in 16 of 35 cases. The morphologic features of aortic body tumors are identical to those of paragangliomas of other locations, and the occurrence of invasive growth and/or metastasis cannot be predicted on histologic grounds.


Primary linitis plastica of the colon: report of two cases and review of the literature. Two cases of primary linitis plastica of the large intestine will be presented and discussed. Characteristic clinical features of this type of adenocarcinoma of the large intestine include younger age of presentation, low incidence of hepatic metastasis and a high incidence of ovarian, lymph node and peritoneal metastasis. Pathological features reveal thickening of the intestinal wall by diffuse infiltrating tumor cells of the characteristic signet ring cells, abortive glands and undifferentiated or anaplastic cells. The prognosis is poor.


Testicular neoplasms in Kenyan Africans. Over a 9-year period 40 testicular and paratesticular neoplasms were seen at the Kenyatta National Hospital, Nairobi, Kenya. Their incidence rate was 0.08 per annum per 100,000 Kenyan males. This low incidence was largely accounted for by a decrease in tumors of germ cell origin. The proportional distribution of the testicular neoplasms, however, was not significantly different from findings in the United States. An inheritable factor apparently controls the decreased susceptability to testicular neoplasms.


Hepatocarcinogenicity of polychlorinated terphenyl (PCT) in ICR mice and its enhancement by hexachlorobenzene (HCB). Studies were made on the carcinogenic effects of hexachlorobenzene (HCB) and polychlorinated terphenyl (PCT) singly and in combination when given orally to mice for 24 weeks. PCT alone induced liver tumors, nodular hyperplasias and hepatocellular carcinomas. HCB alone had no effect, but it enhanced the induction of liver tumors by PCT.


Effects of culture conditions on the growth and differentiation of transformed rat adrenocortical cells. Normal rat adrenocortical cells grow in vitro in two states of differentiation, depending on culture conditions. Under one set of conditions, they proliferate and are fibroblast shaped, with some myoid cell characteristics and with limited steroidogenic activity. Under alternate conditions they do not replicate and are epithelial-like and similar to adrenocortical secretory cells structurally and steroidogenically. The effects of oncogenic transformation on these cellular responses to culture variables were examined by using the Kirsten murine sarcoma virus-transformed rat adrenocortical line TRA. Transformation eliminated the differential proliferative response of the cells to culture variables and resulted in similar growth rates and high cell densities under all conditions. Horse serum, conducive to epithelial-like growth of nontransformed cells, caused aggregation, whereas fetal calf serum, conducive to fibroblastic growth of untransformed cells, caused dispersed growth in TRA cultures. Variations in dissociation procedures had little effect on growth patterns. Most ultrastructural characteristics of myoid as well as adrenocoritcal differentiation were reduced in TRA cells, although basement membranes and extracellular matrix were produced, but under different conditions than in untransformed cultures. Like normal adrenocortical cells, TRA cells were capable of 20alpha-reduction and delta5-3-beta-dehydrogenation of [4-14C5pregnenolone but, in contrast to untransformed cells, they did not respond to variables in culture conditions by qualitative changes in pregnenolone metabolism, did not produce deoxycorticosterone or corticosterone, and secreted progesterone, a hormone not recovered from untransformed cultures. TRA cells showed no increase in steroidogenic differentiation in response to those culture environment factors that normally cause modulation of the fibroblast-shaped adrenocortical stem cells to more differentiated steroid-secreting cells.


Comparison of thioredoxin reductases from Novikoff ascites hepatoma cells and normal liver of rats. Adult rat liver contained variant forms of thioredoxin reductase with isoelectric points at pH 4.9 and at approximately pH 4.7 compared to pH 5.1 for the enzyme from Novikoff ascites hepatoma. Fetal and regenerating liver contained only the form with the isoelectric point at pH 4.9. All three enzymes precipitated with and were inhibited by a rabbit antibody to purified enzyme from Novikoff tumor.


Induction of colonic adenocarcinoma in the rat by X-irradiation. In work to determine whether X-radiation could be used to induce tumors of the colon in outbred Holtzman rats, a technique was devised so that only the descending colon could be irradiated with a collimated X-ray beam and tumorigenic exposures in the kilo-Roentgen range were delivered. Ninety male Holtzman rats were divided into 6 exposure groups of 15 animals each. In the first group only the colon was exposed to 2500 R; in the second group the colon was exposed to 3500 R and the exposures were increased by 1000-R increments up through 6500 R; the sixth group was sham irradiated. Animals in each of the irradited groups developed mucoid adenocarcinomas with the highest incidence (47%) obtained inthe 4500-R group. Histopathologically, the induced tumors bear a close resemblance to human colon adenocarcinoma, and the development of pulmonary metastases in one of the animals with multiple primary tumors demonstrated their malignancy. The usefulness of the animal model is discussed.


Deoxyadenosine antagonism of the antiviral activity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosylhypoxanthine. Deoxyadenosine but not adenosine reversed the antiviral activity of 9-beta-D-arabinofuranosyladenine (ara-A) and 9-beta-D-arabinofuranosylhypoxanthine (ara-H) when used in the presence of coformycin, an inhibitor of adenosine deaminase. In suspension cultures of KB cells, 10 muM ara-A inhibited the replication of herpes simplex virus type 1 by 80%. Concomitant addition of 50 muM deoxyadenosine reduced the antiviral activity of 10 muM ara-A to only 40% inhibition. Adenosine failed to antagonize the antiviral activity. In monolayer cultures of KB cells, the 50% inhibitory concentration of ara-A was increased from 1.5 to 2.9 muM by 2 muM deoxyadenosine and to 8.5 muM by 10 muM deoxyadenosine. Analysis of the dose-response data by a double reciprocal plot method indicated that the antagonism was competitive. The antiviral activity of ara-H also was antagonized by deoxyadenosine. The 50% inhibitory concentration of ara-H was increased from 42 muM to 70, 91, or 121 muM by the concurrent addition of 5, 10, or 20 muM deoxyadenosine. Competitive antagonism could not be demonstrated. In the absence of the adenosine deaminase inhibitor, neither ara-A nor ara-H was antagonized by deoxyadenosine. Since such inhibitors were not available unitl recently, previous investigators were unable to observe the antagonistic capacity of deoxyadenosine.


Isolation and characterization of a cytosol protein (64/7.2) present in large amounts in rapidly growing hepatomas. Protein 64/7.2 (molecular weight/isoelectric point) is present in the cytosol of several hepatomas including the Novikoff hepatoma and Morris hepatomas 9618A, 7794A, and 3924A, but it is not present in liver or 18-hr regenerating liver. Quantitatively, its concentration was highest in Novikoff hepatoma (150 microgram/g tissue) and Morris hepatoma 3924A (550 microgram/g tissue), which are rapid-growing tumors, less in Morris hepatoma 7794A (72 microgram/g tissue), which is of intermediate growth rate, and least in the slow-growing Morris hepatoma 9618A (25 microgram/g tissue). Protein 64/7.2 was isolated from Novikoff hepatoma ascites cells in high purity as shown by its migration as a single band on one-dimensional acid-urea-polyacrylamide gels and a single spot on two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gels. Its amino acid composition has an acidic to basic amino acid ratio of 1.6. Its amino-terminal amino acid is lysine, and its carboxyl-terminal amino acid is glycine. Interestingly, the amino acid composition is strikingly similar to that of the phosphorylated Novikoff hepatoma chromatin Protein Cg', partially characterized in our laboratory.


Morphology and metastatic nature of induced hepatic nodular lesions in C57BL x C3H F1 mice. The metastatic capabilities of well-defined nodular hepatic lesions induced by benzo(a)pyrene, ethylnitrosourea, benzidine.2HCl, and diethylnitrosamine were evaluated. Coded liver and lung tissues from 1264 treated C57BL/6J x C3HeB/FeJ F1 mice were assessed independently for the presence of primary nodular lesions and metastases, respectively. Primary lesions were classified according to their size, cell morphology, and growth patterns into hyperplastic, adenomatous, and trabecular nodules. None of the 126 mice bearing hyperplastic nodules had pulmonary metastases. Four of 291 (1.4%) mice with adenomatous nodular lesions showed metastases. In contrast, of the 733 mice bearing the trabecular type of nodular lesions alone or in combination with other lesions 266 (36%) showed pulmonary metastases. The pulmonary metastases were first detected in mice dying between 51 and 60 weeks of age (5%). This rate increased as a function of age at death, reaching an incidence of 51% in mice surviving more than 81 weeks. It was concluded that nodules showing trabecular and the more anaplastic solid sheet type of growths represented bona fide hepatocellular carcinomas in the mouse.


A comparison of polysomal messenger ribonucleoprotein particles from normal and neoplastic rat liver. Free polysomes were isolated from normal and regenerating rat liver and from Morris hepatomas 7777, 7800, 5123C and 9618A. Sucrose gradient analysis ruled out the possibility of any significant messenger RNA degradation in these polysome preparations. The ethylenediaminetetraacetate-disrupted polysomes were fractionated on oligodeoxythymidylic acid-cellulose columns. The column-bound polyriboadenylic acid-containing messenger ribonucleoprotein particles were eluted with a formamide buffer, precipitated with ethanol, and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The messenger RNA-associated proteins from the different tissues were qualitatively similar, but two proteins with molecular weights of 66,000 and 109,000 found as minor proteins in normal liver appeared on gels as major protein bands when hepatoma messenger ribonucleoprotein particles were examined. The 66,000- and 109,000-molecular-weight proteins in these particles from regenerating liver appeared quantitatively similar to those isolated from normal liver.


Detection of inapparent nodule-transformed cells in the mammary gland tissues of virgin female BALB/cfC3H mice. Inapparent nodule-transformed cells were recovered from morphologically normal mammary tissue from virgin female BALB/cfC3H/Crgl (mouse mammary tumor-positive) mice before the appearance of hyperplastic alveolar nodules (HAN) or tumors, by means of the cell dissociation technique. Mammary tissues were dissociated by means of collagenase (0.1%), hyaluronidase (0.1%), and pronase (1.25%). Aliquots of 10(5) viable cells in 0.01 ml medium were injected into the gland-free mammary fat pads of 3-week-old female syngeneic mice. After 10 weeks the outgrowths were examined and classified as ductal, HAN, tumor, or combinations of these types. The presence of HAN outgrowths indicated the presence of nodule-transformed cells in the donor mammary tissues. Nodule-transformed cells were recovered from 2-month-old donors, and the number of HAN outgrowths increased with donor age. Overt HAN and tumors did not appear in virgin female BALB/cfC3H/Crgl mice younger than 8 to 9 months of age. The data suggest that inapparent nodule-transformed cells occurred long before the appearance of HAN of tumors and that the numbers of transformed cells increased with donor age.


Tumorigenic action of streptozotocin on the pancreas and kidney in male Wistar rats. Pancreatic islet cell tumors were induced in 38 of 44 male Wistar rats (86%), which survived 9 to 14 months following the various treatment schedules. A single i.v. injection of streptozotocin alone, 30, 40, 50, or 65 mg/kg of body weight produced adenomas of pancreatic islet cells in 8 of 9 (89%), 6 of 7 (86%), 2 of 4 (50%), and 1 of 2 rats (50%), respectively. The neoplasms were seen in all of the 8 rats given a single i.p. injection of picolinamide, 250 mg/kg of body weight, 15 min before a single i.v. injection of streptozotocin, 65 mg/kg of body weight. Among the 14 rats given a single i.p. injection of nicotinamide, 500 mg/kg of body weight, 15 min before a single i.v. injection of streptozotocin, 65 mg/kg of body weight, 13 rats (95%) developed pancreatic islet cell tumors. Renal tumors were seen in only 3 rats treated with streptozotocin and nicotinamide. None of rats used in this study developed hepatic tumors. This study demonstrates that steptozotocin, even in a dose of 30 mg/kg of body weight, has an exceedingly marked tumorigenic action on the rat pancreas, while it has little effect on the kidney and no effect on the liver.


Carcinostatic effect of aliphatic aldehydes and aldehyde dehydrogenase activity in Ehrlich carcinoma, Sarcoma 180, and Yoshida AH 130 hepatoma. The antitumor activity of 2,3-dihydroxybutyraldehyde on Ehrlich carcinoma, Sarcoma 180, and Yoshida AH 130 hepatoma, as well as the aldehyde dehydrogenase activity in these tumors, was studied. 2,3-Dihydroxybutyraldehyde at nontoxic doses (500 mg/kg body weight i.p. daily for 7 days) slowed down the growth of solid and ascites tumors in mice. The treatment completely prevented the development of Yoshida ascites hepatoma in several rats. 2,3-Dihydroxybutyraldehyde, although it did not influence the growth of Ehrlich carcinoma transplanted in the brain of mice, significantly decreased in the lungs of these animals the number of viable tumour cells that derived from the primary tumor. All the tested tumors, which were sensitive to the action of 2,3-dihydroxybutyraldehyde, were virtually devoid of aldehyde dehydrogenase activity. These results suggest a possible relationship between the lack of this enzyme activity and the antitumor activity of aliphatic aldehydes.


Influence of dietary protein and vitamin B12 on the toxicity and carcinogenicity of aflatoxins in rat liver. The influence of dietary protein content and dietary vitamin B12 supplement on the hepatotoxicity and carcinogenicity of aflatoxin in rat liver was studied. In animals fed a low-protein diet, aflatoxin induced extensive toxic and carcinogenic effects. Cirrhosis was significantly prevented to a certain level by vitamin B12 administration, but the incidence of cholangiofibrosis and hyperplastic nodules was unchanged. No toxic effect was observed in animals receiving high-protein diet with no vitamin B12 supplement in this study (33 weeks). Only one rat bearing a hepatoma was observed in this group. However, hepatoma and hyperplastic nodules were found in the group receiving high-protein diet plus vitamin B12. Cholangiofibrosis and cirrhosis were not observed in the high-protein group regardless of vitamin B12 administration.


Peripheral neuropathy as a complication of cis-dichlorodiammineplatinum(II) treatment: a case report. A 19-year-old woman with a diagnosis of osteosarcoma was initially treated with amputation of her right leg and adjuvant adriamycin. She developed pulmonary metastases 18 months following diagnosis. She was then given cis-dichlorodiammineplatinum(II) (DDP) at a dose of 100 mg/m2 iv approximately every 4 weeks as the sole drug. Following the fifth dose of DDP, she complained of numbness and tingling in her hands and leg. A distal sensory loss extending to both elbows and her remaining knee was found on examination. Nerve conduction tests were compatible with peripheral neuropathy of the "glove and stocking" type. DDP was withheld and her sensory loss improved over the next 2 months, but became worse after another course of DDP was administered. The temporal relationship between the findings and the administration of DDP implicates this drug as the causative agent in the peripheral neuropathy.


Investigations into the mechanism of hormone release by rat adrenocortical cells. Vinblastine treatment blocks corticosterone release from rat adrenal zona fasciculata without impairing hormone synthesis, and induces the formation of acid phosphatase-positive granular clumps at the juxta-sinusoidal pole of the cells. Autoradiography shows that ACTH administration to vinblastine-treated animals mobilizes the 3H-cholesterol stored in the lipid droplets and leads to a noticeable labelling of the granular clumps. The possible significance of these granules is discussed.


Introduction of the herpes simplex virus thymidine kinase gene into mouse cells using virus DNA or transformed cell DNA. Cells lacking the enzyme thymidine kinase (LMTK- cells) have been transformed to a kinase-positive phenotype using sheared herpes simplex virus (HSV) DNA, and the enzyme found in these transformed cells is HSV-specific. One of the cell lines is able to complement the functional defect found in two temperature-sensitive mutants of HSV 1, and reversion of the cells to a thymidine kinase-negative phenotype results in the loss of this capability. The HSV thymidine kinase gene can also be introduced into LMTK- cells using DNA extracted from transformed cells, and the high efficiency of this procedure suggests that the state of the virus DNA in transformed cells is different from that of DNA in virus particles.


Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistnat CHO cells. The carbohydrate moieties of the G glycoprotein of vesicular stomatitis virus (VSV) grown in three distinct lectin-resistant (LecR) Chinese hamster ovary (CHO) cell lines have been compared by fine structural analysis of radiolabeled glycopeptides. The mutant WgaRIII, selected for resistance to wheat germ agglutinin (WGA), produces VSV containing G glycoprotein specifically lacking in sialic acid. The mutant PhaRI, selected for resistance to phytohemagglutinin (PHA) and previously shown to lack a particular glycoprotein N-acetyl-glucosaminyl-transferase activity, produces VSV containing G glycoprotein specifically lacking terminal N-acetylglucosamine-galactose-sialic acid sequences and possessing an increased number of mannose residues in the "core" region of its carbohydrate moieties. The mutant PhaRIConARII, a "double" mutant selected from PhaRI cells for resistance to concanavalin A (ConA), produces VSV containing G glycoprotein with a further alteration in the mannose residues of the "core" oligosaccharide region. We discuss the relevance of these findings to the mechanisms of glycoprotein biosynthesis in mammalian cells and to the biochemical bases of lectin resistance in CHO cells.


Effects of tropic hormones on ultrastructure and synthesis of androgens in adrenals of male pseudohermaphrodite rats. Ultrastructural and biochemical study of the adrenals in the pseudohermaphrodite (tfm) rat reveals hypertrophic adrenocortical cells. The cytoplasm of the cells in the zonae fasciculata and reticularis contains an exceptionally well developed smooth endoplasmic reticulum closely applied to mitochondria and lipid droplets. Mitochondria are more numerous than in normals and have especially abundant tubular cristae. More lipid droplets (appearing as empty vacuoles) are surrounded by pleomorphic mitochondria. The incubation study indicates that the capacity of rat adrenal cortex of producing androgens is greater in tfm than in normal animals. Hypophysectomy and castration result in a significant decrease in androgen biosynthesis by tfm rat adrenals and cause a reduced concentration of plasma testosterone. Administration of tropic hormones to hypophysectomized-castrated rats appears to stimulate their adrenal androgenesis. It is suggested that in tfm rats the higher than normal luteinizing hormone (LH) together with adrenocorticotropic hormone (ACTH) stimulates the hypertrophy of cellular organelles in the adrenal cortex and causes an accompanying increase in androgenic steroids which may be responsible, at least in part, for the increased level of plasma androgens.


Identification of a transformation-specific protein induced by a Rous sarcoma virus. Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.


Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil. When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.


Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein. The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.


Avian myelocytomatosis and erythroblastosis viruses lack the transforming gene src of avian sarcoma viruses. Using labeled cDNA specific for the detection of the src gene of avian sarcoma viruses, we find that avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus strain ES4 lack nucleotide sequences related to the src gene. Furthermore, chicken fibroblasts as well as hematopoietic cells, infected and transformed with these viruses, show no enhanced level of transcription of the cellular nucleotide sequences related to the src gene of avian sarcoma viruses. These two viruses may thus contain their own transforming gene(s) or induce cellular genes unrelated to the src-like cellular sequences.


MAC-1, a new genetically transmitted type C virus of primates: "low frequency" activation from stumptail monkey cell cultures. A new class of endogenous primate type C virus has been isolated from a continuous tissue culture line of Macaca arctoides cells by co-cultivation with a human cell line. The virus, designated MAC-1, can be transmitted to human and feline cells in tissue culture, and is unrelated, by immunological and nucleic acid hybridization criteria, to previously characterized retroviral isolates of primates. In particular, MAC-1 shows no detectable homology to the baboon type C viruses, even though viral genes related to the latter group are readily detected in M. arctoides cellular DNA. Viral gene sequences related to the MAC-1 genome are present in multiple copies (50-150 per haploid genome) in Old World primates, and are expressed in the cellular RNAs of uninfected and "virus-free" primate cells and tissues. Thus there are at least two distinct sets of genetically transmitted Old World primate type C viral genes, each of which is found in multiple copies in normal primate cellular DNA. With the description of this new retrovirus, there are now a minimum of five distinct genetically transmitted viruses of primates, three type C and type D, each represented in multiple copies in the normal cellular DNA.


Electron microscopic evidence for splicing of SV40 late mRNAs. Poly(A)-containing SV40 cytoplasmic RNA was hybridized with linear double-stranded SV40 DNA and formed RNA displacement loops (R loops). The structures visualized in the electron microscope are consistent with the conclusion that the leader sequences at the 5' ends of the 16S and 19S late mRNAs are not coded immediately adjacent to the main portions of the mRNAs. These data are consistent with either segmentation of the leaders or heterogeneity of their lengths. Measurements carried out on the R loop structures have provided the locations, on the physical map of SV40 DNA, for the bodies and leaders of the 16S and 19S late mRNAs, and the lengths of the bodies, leaders and the corresponding intervening DNA sequences.


Construction of a viable SV40 variant containing two functional origins of DNA replication. Viable variants of simian virus 40 (SV40) have been constructed which contain two functional origins of DNA replication (Or). The variants were made by introducing, at 0.175 on the SV40 map, a segment of DNA containing the viral Or. Two types of experiments demonstrate that the second Or is functional. First, the distribution of radioactivity in pulse-labeled SV40 (I) DNA is dramatically altered in the variants when compared with the parental virus. Second, electron microscopic examination of viral replicative intermediates indicates that while there is one initiation site for DNA synthesis in the parental genome, there are two sites in the variant. It was possible to introduce a deletion which inactivated the original Or at 0.67 map units in this variant. The resulting mutant could be propagated,  and its DNA replication originated at the site of the newly inserted Or.


Effects of BUdR on developmental functions of Dictyostelium discoideum. The development of Dictyostelium discoideum cells, as measured by spore yield, is somewhat more sensitive to the presence of BUdR during vegetative growth than is growth itself. Observations on the development of control and BUdR-grown cells, their protein labelling patterns and assays of 4 developmentally regulated proteins all reveal a consistent picture. BUdR appears to block spore formation by partially inhibiting several or many different earlier events during development. The relative sensitivity of development compared to growth to inhibition by the drug may be a consequence of the nature of the developmental process rather than of some unique specificity of the inhibitor.


Resurgence of glycogen synthesis and storage capacity in cultured hepatoma cells. This paper describes a new cultured hepatoma cell line referred as ZHC cells, derived from the ascitic Zajdela rat hepatoma. Since 1963, the dedifferenciated in vivo transplanted ascitic cells were characterized by the absence of glycogen as in generally the case in all fast growing hepatic tumors. In 1974, we succeeded in adapting these tumor cell to in vitro defined growth conditions, where we observed the progressive recovery of the ability to synthesize and to store large amounts of glycogen, as shown by histochemical, ultrastructural and biochemical studies. It can now be considered as an established cell line in which the reverted phenotype has been stable for 3 years.