Hydrolytic action of phospholipases on bacterial membranes. Substrate specificities of phospholipases C[EC 3.1.4.3] from Clostridium novyi, Clostridium perfringens, Bacillus cereus, and Pseudomonas aureofaciens were studied under the same conditions. Phospholipases C from Clostridium novyi and Bacillus cereus show wide substrate specificities while those of Clostridium perfringens and Pseudomonas aureofaciens show relatively narrow specificities. On the basis of these results, the hydrolytic actions of these phospholipases on membrane lipids of Escherichia coli, Bacillus cereus, and Clostridium novyi were examined under the same conditions. The enzymes of Clostridium novyi and Bacillus cereus attacked all the membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine, phosphatidylglycerol, lyso-phosphatidylethanolamine, and o-aminoacylphosphatidylglycerol. Phospholipase C from Pseudomonas aureofaciens attacked these three membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine. Phospholipase C from Clostridium perfringens hardly attacked the phospholipids of these bacterial membranes. However, phospholipase C from Clostridium perfringens hydrolyzed phosphatidylethanolamine in a mixture containing lipid extract from Escherichia coli membrane and purified phosphatidylcholine from egg yolk.


On the reaction of cyanide with an oxygenated form of cytochrome oxidase. The reaction of an oxygenated form of cytochrome oxidase [EC 1.9.3.1] with cyanide was examined under conditions where spontaneous decay was prevented. The equilibrium and kinetic constants for the reaction agreed well with those for the normally operating enzyme, indicating that the oxygenated form is one of the active intermediates of the cytochrome oxidase reaction.


Degradation of benzenesulfonate to sulfite in bacterial extract. Sulfite formation from benzenesulfonate was studied in extracts from a bacterium grown on this compound as a main carbon source. The activity of sulfite formation depended on the presence of NADH and oxygen as well as magnesium, suggesting an oxygenation-type reaction. The activity was found in a fraction precipitated by ammonium sulfate at 35-50% saturation; the specific activity was 15 times higher than that of crude extract, probably due to the elimination of inhibitory substances of low molecular weight from the preparation. In a distillate of the reaction mixture, phenol was found. Pyrocatechol as well as benzenesulfonate was oxidized in the crude extract, but phenol was not.


1-Methoxy-5-methylphenazinium methyl sulfate. A photochemically stable electron mediator between NADH and various electron acceptors. This paper describes the properties and application of 1-methoxy-5-methylphenazinium methyl sulfate (1-methoxyPMS), which is a photochemically stable, versatile electron carrier. Like 5-methylphenazinium methyl sulfate (PMS), it mediates electron transfer between NADH and various electron acceptors such as tetrazolium dyes or the electrode of an enzymic electric cell, and yet it does not deteriorate upon storage under scattered light in normal laboratories. The rate of reduction of 1-methoxyPMS coupled to the reoxidation of NADH produced by the lactate dehydrogenase reaction, was even faster than that of PMS. It was also successfully employed as an electron mediator in the enzymic electric cell method for the assay of NAD-linked dehydrogenases. 1-MethoxyPMS solution is rosy pink, and its standard redox potential (E0') is approximately +0.063 V. The use of 1-methoxyPMS will be beneficial in biochemistry as well as medical technology, where PMS has been used as an electron mediator in various electron transfer systems.


Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99. Sensitive thin layer peptide mapping is employed to establish the identity and the homogeneity of eight singly substituted 4-carboxy-2,6-dinitrophenyl derivatives of horse cytochrome c. Seven of the components, all of greater than 95% homogeneity, are modified at lysyl residues 13, 72, 87, 8, 27, 39, and 60. The eighth component is a mixture of derivatives at lysines 22 and 99. The positions of the modified residues were confirmed by the amino acid analysis and Edman sequential degradation of the CDNP-peptides. Physiochemical properties characteristic of cytochrome c are unchanged in the chemically modified products examined. These properties, that include the proton NMR spectrum, are sensitive probes of the polypeptide organization surrounding the heme prosthetic group. The lack of any discernable changes indicates that modification of the epsilon-amino groups on the surface of cytochrome c does not perturb the overall structure of the protein. The widespread distribution of the modifications on the surface of the molecule, together with the homogeneity and native conformation of the CDNP-derivatives make them well suited for assessing the effects of changes in the charge topography on the electron transfer activity of cytochrome c.


Influence of potassium salt concentration and temperature on inhibition of mRNA translation by 7-methylguanosine5'-monophosphate. The effect of 7-methylguanosine 5'-monophosphate (pm7G) on mRNA translation was examined in the wheat germ and rabbit reticulocyte cell-free systems. Differences between the two cell extracts with respect to inhibition of translation by pm7G can be attributed to different conditions commonly used for in vitro protein synthesis. Inhibition of globin mRNA translation by pm7G is strongly influenced by the concentration of potassium salt and to a lesser extent by incubation temperature. The effectiveness of the inhibitor increases with potassium salt concentration and diminishes with increasing temperature. Translation is inhibited by pm7G at physiological K+ concentration in both cell-free systems in that only the rate of binding of mRNA to ribosomes is affected by the inhibitor, not the extent of binding. Translation of different capped mRNAs is affected differently by pm7G, but this appears to be property of the mRNA rather than the translation system. These results indicate that while the 5'-terminal cap structure may be more important for translation of some mRNA's than others, this structure functions in translation of capped mRNAs in all types of cells.


Properties of 2-nitropropane dioxygenase of Hansenula mrakii. Formation and participation of superoxide. 2-Nitropropane dioxygenase, purified to homogeneity from a yeast, Hansenula mrakii, is significantly inhibited by superoxide dismutase and various scavengers for superoxide anion such as cytochrome c, epinephrine, NADH, thiols, and polyhydric phenols. The reduction of cytochrome c and the oxidation of epinephrine and NADH are concomitant with the inhibition of enzymatic oxygenation. Neither the oxidation nor the reduction occursin the presence of superoxide dismutase or in the absence of 2-nitropropane or oxygen. Superoxide anion added externally induces the oxygenation. These findings indicate the generation of superoxide anion and its participation in the oxygenation of 2-nitropropane. The difference spectrum of the binding of NADH to 2-nitropropane dioxygenase exhibits a negative peak at 353 nm. One mole of NADH is bound to 1 mol of the enzyme and the pro-R hydrogen of the nicotinamide moiety of bound NADH predominantly is transferred to superoxide anion formed enzymatically or given externally. Thus, the diastereotopic hydrogen of NADH is discriminated by the enzyme, although not completely.


Heme is necessary for the accumulation and assembly of cytochrome c oxidase subunits in Saccharomyces cerevisiae. The presence of cytochrome c oxidase subunits and the association of these subunits with each other was studied in a heme-deficient Saccharomyces cerevisiae mutant. This mutant had been isolated by Gollub et al. (1977) J. Biol. Chem. 252, 2846-2854) and had been shown lack delta-aminolevulinic acid synthetase. When grown in the absence of heme or heme precursors, the mutant is respiration-deficient, devoid of cytochrome absorption bands and auxotrophic for all those components whose biosynthesis is dependent on hemoproteins; when grown in the presence of heme or heme precursors, the mutant is phenotypically wild type. Upon growth of the mutant in the absence of heme synthesis, the mitochondria still contained two of the three mitochondrially made cytochrome c oxidase subunits (i.e. II and III) and at least one of the cytoplasmically made cytochrome c subunits (VI). The other subunits were either barely detectable (I, IV) or undetectable (V, VII). The residual subunits were apparently not assembled with each other since an antiserum directed mainly against Subunit VI failed to co-precipitate Subunits II and III which were still present. In contrast, growth of the mutant in the presence of delta-aminolevulinic acid led to the accumulation of active, fully assembled cytochrome c oxidase in the mitochondria. Heme a (or one of its precursors) thus controls the assembly of cytochrome c oxidase from its individual subunits.


Evidence for structural homology between human red cell phosphoglycerate mutase and 2,3-bisphosphoglycerate synthase. Previous reports have suggested the possibility of extensive structural homology between human erythrocyte bisphosphoglycerate synthase (glycerate-1,3-P2 leads to glycerate-2,3-P2) and phosphoglycerate mutase (glycerate-3-P in equilibrium glycerate-2-P). This study lends credence to that conjecture through comparative physicochemical investigations involving peptide mapping, circular dichroism, and immunological techniques. The data indicate that despite differences in function, both enzymes apparently manifest a high degree of similarity in primary, secondary, and tertiary structure. Mapping data also indicate that each protein is comprised of two apparently identical subunits.


Phosphorylation of cardiac troponin by guanosine 3':5'-monophosphate-dependent protein kinase. Homogeneous cGMP-dependent protein kinase catalyzes the rapid incorporation of phosphate, specifically into the inhibitory subunit of purified cardiac troponin with a maximal incorporation of 1 mol of phosphate/mol of troponin. When troponin was incubated in the presence of both cGMP- and cAMP-dependent protein kinases, a maximal incorporation of 1 mol of phosphate/mol of troponin was observed which suggested phosphorylation of the same site by the two kinases. Both cyclic nucleotide-dependent kinases had similar Km values for troponin, but the Vmax value for the phosphorylation reaction catalyzed by cAMP-dependent protein kinase was 12-fold greater than the value obtained for cGMP-dependent protein kinase.


Purified cyclic GMP-dependent protein kinase catalyzes the phosphorylation of cardiac troponin inhibitory subunit (TN-1). Cyclic AMP- and cGMP-dependent protein kinases catalyze the phosphorylation of cardiac troponin inhibitory subunit (TN-I). Unlike many substrates utilized by both kinases, TN-I is rapidly phosphorylated using relatively low concentrations of the cGMP-dependent protein kinase (0.01 to 0.1 micrometer). At low concentrations of cAMP- and cGMP-dependent protein kinases, approximately twice as much total phosphate is incorporated into TN-I using the cAMP-dependent enzyme. At higher enzyme concentrations, 1 mol of phosphate/mol of TN-I is found using either enzyme. Maximal levels of cAMP- and CGMP-dependent protein kinases do not catalyze additive phosphorylation, suggesting that the two enzymes catalyze the phosphorylation of the same site on TN-I. The results support the concept of overlapping substrate specificity for cAMP- and cGMP-dependent protein kinases, but suggest that cardiac troponin contains additional specificity determinants for the cGMP-dependent protein kinase not found in several other protein substrates.


Sequence homology of the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis with other Ca2+-binding proteins. A Ca2+-dependent regulator protein of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+-binding protein with a molecular weight of approximately 17,000. The Ca2+-dependent regulator protein is also structurally similar to troponin-C, the Ca2+-binding component of muscle troponin and Ca2+ mediator of muscle contraction. The present report describes a partial amino acid sequence of the Ca2+-dependent regulator. The protein (148 amino acids) is 50% homologous with skeletal muscle troponin-C, but is 11 residues shorter than the muscle protein. The Ca2+-dependent regulator protein has an NH2-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of epsilon-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca2+-binding proteins. These properties may account for the biological specificities demonstrated by these proteins as compared to the Ca2+-dependent regulator protein. Based on the sequence and a comparison of the Ca2+-dependent regulator protein to other calcium-binding proteins, our data support the view that all of these moecules contain common sequences, especially at their proposed metal-binding sites.


Effect of pressure and ionic strength on the self-association of Apo-A-I from the human high density lipoprotein complex. The self-association of apo-A-I isolated from the human high density lipoprotein complex has been investigated by gel permeation chromatography and sedimentation equilibrium. The apparent weight average molecular weight (MWapp) versus Apo-A-I concentration profile was found to be sensitive to ionic strength and pressure; MWapp increased with increasing ionic strength and decreasing rotor speed. The data were consistent with a monomer-dimer-tetrameroctamer association shceme over all conditions investigated if a change in the partial specific volume of apo-A-I upon association of 5.5 x 10(-2) ml/g is postulated.


Phosphodiesterase activator from rat kidney cortex. Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.


Transport of a nonphosphorylated nucleoside, 5'-deoxyadenosine, by murine leukemia L1210 cells. The mode of transport of a nonphosphorylated adenosine analog, 5'-deoxyadenosine, was studied in murine leukemia L1210 cells. This compound is not subject to the action of intracellular nucleoside-trapping kinases, and its transport can be examined without regard for effects of experimental conditions on kinase activity. Accumulation of 5'-deoxyadenosine was rapid, and nonconcentrative, with equilibrium attained within 12 s at 37 degrees. Kinetic studies were carried out at 20 degrees. We found both a nonmediated (diffusion) and a mediated transport process. The latter had an apparent Km fo 115 micrometer, Vmax = 105 pmol/10(6) cells/min. Uptake of 5'-deoxyadenosine was inhibited by several heterologous nucleosides including adenosine, 2'-deoxyadenosine, thymine riboside, and inosine. Like 2'-deoxyadenosine, 5'-deoxyadenosine was more lipid-soluble than adenosine (from octanol/water partition studies). Compared with 5'-deoxyadenosine, adenosine had a much lower apparent Km (5 micrometer) and a higher Q10 over the 27-37 degrees range (3.0 versus 1.3). Data obtained with adenosine might, however, reflect properties of intracellular adenosine kinase interacting with a transport process.


Synthesis of photopigments and electron transport components in synchronous phototrophic cultures of Rhodopseudomonas sphaeroides. The kinetics of synthesis and incorporation of the photosynthetic pigments and several of the major oxidative and photosynthetic electron transport components of Rhodopseudomonas sphaeroides have been studied during synchronous and asynchronous phototrophic growth. The photosynthetic pigments and cytochromes c and b, measured spectroscopically, exhibited continuous patterns of synthesis and incorporation into the membrane particulate fraction in both synchronous and asynchronous cultures. Succinic dehydrogenase and NADH-oxidase activities, present at low levelnous growth. In a previous paper, Leuking, D.R., Fraley, R.T., and Kaplan, S. ((1978) J. Biol. Chem. 253, 451-457) have shown that total cellular phospholipid is also accumulated discontinuously during synchronous growth. A continuously incorporated membrane component is thus subject to a wide variation in the membrane protein/lipid ratio. The significance of this ratio in regulating the activity of membrane proteins is discussed and the distinction between protein incorporation and function is drawn with particular reference to the photosynthetic pigments and cytochrome components and the oxidative activities measured. It is suggested that a dependence of membrane protein activity on the membrane protein to lipid ratio in vivo is of possible significance in the control of membrane synthesis and cell division.


Reconstitution of the apoenzyme of cytochrome oxidase from Pseudomonas aeruginosa with heme d1 and other heme groups. Cytochrome oxidase (EC 1.9.3.2) from Pseudomonas aeruginosa contains heme d1 and heme c in an equimolar ratio. The heme d1 can be removed from the enzyme with acidified acetone leaving an apoenzyme that contains heme c but has no oxidase activity. Reconstitution of the apoenzyme in neutral 6 M urea with heme d1 yields a reconstituted product which, after removal of the urea, has 90 to 100% of the oxidase activity of the native enzyme, a 1:1 molar ratio of the heme groups, and is indistinguishable from the native on the basis of its absorption spectral properties and its EPR spectrum. The apoenzyme can also be reconstituted with heme a, deuteroheme, hematoheme, mesoheme, and protoheme but only the heme a yields a product with any oxidase activity. The properties of these reconstituted products are compared.


Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver. A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.


Cellular RNA synthesis in normal and mengovirus-infected L-929 cells. Cellular RNA synthesis was studied in mouse L-929 cells and in these cells infected with mengovirus. RNA polymerases I, II, and III were partially purified and their chromatographic properties were analyzed by DEAE-Sephadex A-25 chromatography. RNA polymerase II was purified from mouse liver and its subunit structure was compared to that of normal and virus-infected L-929 cells by two-dimensional gel electrophoresis. By these criteria, the enzymes from all three sources were identical. The RNA synthetic activities and capacities of chromatins from normal and virus-infected cells were compared under a variety of conditions. The endogenous activity in chromatin from infected cells was inhibited relative to controls but the residual activity responded normally to stimulation by ammonium sulfate, heparin, and Sarkosyl. The template capacity of the chromatins was compared with added RNA polymerase II and by a rifampicin challenge assay utilizing Escherichia coli RNA polymerase. Identical results were obtained in each case. The number of growing RNA chains and the rates of their elongations were determined. The results showed that nuclei and chromatin from infected cells have a smaller number of RNA polymerase II molecules engaged in RNA synthesis than normal cells do but that the active molecules elongate RNA chains at the same rate.


Nucleotide sequence of the DNA encoding the 5'-terminal sequences of simian virus 40 late mRNA. We have used a combination of techniques of DNA and RNA sequence analysis to determine the nucleotide sequence of the portion of simian virus 40 DNA preceding and encoding the 5' end of mRNA for the structural protein VP2 of simian virus 40. Comparison of the sequence with those found in polyadenylated RNA in the cytoplasm of infected cells RNA shows that the transcript of sequences preceding the structural gene is more abundant than the transcript containing the codons for the protein. Between the abundant transcript of sequences preceding the coding region and the less abundant transcript of the coding region there is a short sequence whose transcript is not detected.


Nucleotides sequence of the genes for the simian virus 40 proteins VP2 and VP3. We have determined the nucleotide sequence of the DNA of simian virus 40. The proceeding report (Dhar, R., Reddy, V.B., and Weissman, S.M. (1978) J. Biol. Chem. 253, 612-620) presents the sequence of a portion of the simian virus 40 DNA that overlaps the region encoding the 5' end of the minor structural protein VP2. We report here the sequence of the remainder of the genes for minor structural proteins VP2 and VP3. The results indicate that the mRNA for the two proteins is read in the same phase and the initiation site for VP3 lies within the structural gene of VP2. The codons of the COOH-terminal amino acids of VP2 and VP3 are read in a second phase as the codons of the NH2-terminal amino acids of VP1.


A polymethacrylate-silica composite material for dental implants. A study has been carried out on the structure and surface texture of a new dental implant material composed of silica microspheres (3 or 5 wt%) and poly(methyl methacrylate). A recently developed composite material composed of vitreous carbon microballoons and poly(methyl methacrylate) has proven highly successful in clinical use, but the black color presents aesthetic problems at the gingival margin. The new material was developed in order to reproduce the many desirable qualities of the vitreous carbon-polymethacrylate composite, while omitting the black color. Square wafers (10 mm X 10 mm X1 mm) were studied, with the surface sandblasted in half of the specimens. Light microscopy revealed an even spacing of spherical configurations throughout the material. Scanning electron microscope studies revealed a finely porous surface with many large craters in the sandblasted specimens.


Sites of lipoprotein particles in normal rat hepatocytes. Very low density lipoprotein (VLDL) particles are packaged by the Golgi apparatus into vacuoles which move to the plasma membrane and empty the particles into the space of Disse, via exocytosis. Traditionally, all lipoprotein-containing cisternae and vacuoles are thought to be parts of this pathway. Observations reported here demonstrate that there is a second population of lipoprotein-containing cisternae and vacuoles. This population is part of GERL, an organelle we consider to be a specialized hydrolase-rich region of the endoplasmic reticulum (ER). To our knowledge, this is the first systematic study of GERL in normal rat hepatocytes.


Ultrastructural changes and cyclic AMP in frog oxyntic cells. In vitro frog gastric mucosa was employed as a model for a combined physiological, biochemical, and ultrastructural study of the morphological changes which accompany the onset of acid secretion by the oxyntic cell. The histamine H2-receptor antagonist metiamide was used to provide a reproducible control state. Stimulation of acid production by theophylline resulted in a 10-fold increase in plasma membrane surface area and a distinct change in the conformation of mitochondrial cristae. Studies using the acid secretion inhibitors, thiocyanate and anoxia, demonstrated that neither acid production per se nor oxidative metabolism is essential for the theophylline-dependent changes in surface area. Increases in tissue cyclic AMP levels were observed under the conditions producing morphological changes. It is postulated that surface area changes induced by theophylline are controlled by cellular cyclic AMP levels.


Reversible inhibition of the norepinephrine induction of lactate dehydrogenase by cytochalasin B in rat glial C6 cells. The cyclic AMP mediated induction of lactate dehydrogenase (LDH: E.C. 1.1.1.27) activity by norepinephrine in the rat glial cell line C6 is inhibited by cytochalasin B. Doses of 5, 15, and 25 microgram/ml of cytochalasin B inhibited the induction equally. Twenty-five microgram/ml of cytochalasin B inhibited the induction reversibly, and had no effect on basal enzyme level. No effect of cytochalasin B on general protein synthesis was found, nor did it increase the rate of decline of enzyme activity in deinduced cells. It therefore appears to block LDH induction by selectivity inhibiting its synthesis. Cytochalasin B had no effect on the transient (intracellular and extracelllular) rise in cyclic AMP generated in response to norepinephrine treatment. Cytochalasin B was effective when added during the transcription dependent phase (first 3 hours) but not during the translation dependent phase (after 3 hours) of LDH induction. The suggestion is discussed that cytochalasin B inhibits one of the early events of the inductive process.


The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics. Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock-infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings. The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformants. The SVTER cell lines were tested for creatine phospholinase (CPK), an enzyme activity chracteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies  and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.


Dibutyryl cyclic AMP arrests the growth of cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle. DBcAMP reversibly arrests cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle. This is supported by the measurement of DNA synthesis by autoradiography and measurement of cellular DNA by two methods--the diphenylamine reaction and microspectrophotometry of Feulgen stained cells. We also present evidence that (1) cell division is prevented if DBcAMP is added as late in the cycle as early S phase. (2) The inhibition of cell division does not appear to be caused by products of tyrosine oxidation. (3) The increase in cell size that occurs in the presence of DBcAMP reflects continued synthesis of protein in the absence of cell division.


Effects of cyclic AMP on the growth of differentiating and undifferentiated Friend erythroleukemic cells. Elevated concentrations of cyclic AMP elicit only minor reductions in growth rate and saturation density in undifferentiated Friend erythroleukemic cells. During the course of dimethylsulfoxide (DMSO)-induced differentiation, Friend cells convert from a cyclic AMP-tolerant state to a phenotype characterized by a high degree of sensitivity to cyclic AMP-mediated growth arrest. Conversion to cyclic AMP sensitivity is detectable after 30 hours growth in medium containing 2% DMSO, and either 0.5 mM 8-Br-cyclic AMP or 5 nM cholera toxin. Cultures of differentiating Friend cells achieved a stationary phase density that was approximately 8-fold higher than the cell density observed in parallel, differentiating cultures treated with 0.5 mM 8-Br-cyclic AMP. Temporally, the appearance of cyclic AMP-sensitivity corresponds to the early expression of in vitro erythroid differentiation (Ross et al., '74), but growth arrest does not alter the subsequent accumulation of hemoglobin in non-dividing DMSO-induced cells. Since growth arrest is preceded by a round of cell division, these observations are consistent with the concept that DMSO must be present during DNA replication for the subsequent expression of hemoglobin synthesis (McClintock and Papaconstantinou, '74; Levy et al., '75; Harrison, '76).


The role of butyrate in the reverse transformation reaction in mammalian cells. The reverse transformation reaction of Chinese hamster ovary cells from compact, epithelial-like, randomly growing, heavily knobbed, lectin reactive cells into stretched, tighly adherent, smooth-surfaced, lectin resistant, fibroblast-like cells normally elicited by dibutyryl cAMP can be produced to its complete extent by N6-monobutyryl cAMP or 8-bromo-cAMP, O2'-monobutyryl cAMP is ineffective as is cAMP itself in the absence of an inhibitor of phosphodiesterase activity. In the presence of a phosphodiesterase inhibitor, cAMP is fully effective. These results indicate that the role of the butyryl groups of dibutyryl cAMP and, especially, the N6-butyryl, in the reverse transformation reaction is protection of the cAMP analogue from degradation. Butyrate at concentrations of about 1 mM does produce a response which to some extent mimics that of cAMP analogues. The cells, however, fail to assume a fibroblastic-like shape, but rather become flattened. The butyrate effect is much slower and less readily reversible than that evoked by cAMP analogues. Butyrate produces an approximately 2-fold increase in intracellular cAMP levels. These results are consistent with the hypothesis that butyrate effects, in part, are mediated by AMP.


Oscillations and cell development in Dictyostelium discoideum stimulated by folic acid pulses. Folic acid is known to be a chemoattractant of pre-aggregation cells of Dictyostelium discoideum. When supplied in pulses, folic acid induces biochemical oscillations and stimulates the development of pre-aggregation to aggregation-competent amoebae. The continuous supply of folic acid has no stimulatory effect. Folic-acid-induced oscillations are accompanied by periodic changes in the cyclic AMP concentration. Pulses of folic acid applied with rhythms between 7 and 11 min efficiently induce oscillations. In contrast, a rhythm of 2 min neither induces oscillations nor suppresses them. Cells start to oscillate with a rhythm of about 8 min. This inherent rhythm is independent of the inducing rhythm. Oscillating cells are less sensitive to folic acid than pre-oscillating ones. They respond only to high concentrations of folic acid which also interact with the oscillating system.


Role of Ca2+ in preserving viability of Friend erythroleukaemic cells after ultramicroinjection. Ca2+ facilitated the fusion by Sendai virus of Friend erythroleukaemic cells and Ehrlich ascites tumour cells but not that of hepatoma tissue culture cells. In the absence of Ca2+ Sendai virus caused the complete depletion of ATP and abolished protein synthesis in Friend erythro-leukaemic cells fused with each other. Addition of high concentrations of Ca2+ (10-20 mM) partially protected the cells from ATP depletion. After a further incubation of cells in complete medium plus 0.2 mM adenine, ATP levels and protein synthesis were restored to 60-85% of those of the untreated control. The protective effect of Ca2+ was used to improve the ultramicroinjection method which involves the fusion of human erythrocyte ghosts with cells. When human erythrocyte ghosts containing high K+ were fused with Friend erythroleukaemic cells in the presence of 10 mM Ca2+ ATP levels and protein synthesis after recovery were about 60-85% of the control. Friend erythroleukaemic cells subjected to ultramicroinjection under these conditions had a cloning efficiency of 75-95% of that of the untreated controls. In these experiments 70-100% of the cells had fused with ghosts. Induction of haemoglobin synthesis by dimethylsulphoxide was unimpaired in cells subjected to ultramicroinjection under the same conditions.


Dynamics and characterization of plasma immunoreactive beta-melanocyte stimulating hormone in hemodialysis patients: its relationship to ACTH. We studied the plasma immunoreactive beta-MSH ("beta-MSH") in hemodialysis patients to determine its basal level, plasma disappearance rate, gel filtration and immunological characteristics. All patients had increased plasma "beta-MSH" (90--440 pg/ml; normal less than 90 pg/ml). Plasma ACTH and cortisol values were within the normal range. Cortisol infusion over 2 h induced almost no plasma "beta-MSH" variation as compared to controls where "beta-MSH" decreased rapidly (apparent half-life 90 min.); more prolonged administration of corticosteroids (dexamethasone 0.5 mg every 6 h for two days) caused a slight (20%) but significant (P less than 0.001) decrease of "beta-MSH." On Sephadex G-50 endogenous "beta-MSH" eluted in a molecular weight range of 6,000--10,000. In our radioimmunoassay dilution curves of endogenous "beta-MSH" paralleled that of synthetic human beta-MSH, but not that of purified human beta-LPH. In conclusion, hemodialysis patients show a clear dissociation between elevated "beta-MSH" and normal ACTH plasma levels. "beta-MSH" probably has a decreased plasma disappearance rate and seems related to a substance different from human beta-MSH.


Cure of Cushing's disease by transsphenoidal removal of a microadenoma from a pituitary gland despite a radiographically normal sella turcica. A 37-year-old woman had symptoms of Cushing's disease for two years. Galactorrhea was present. The diagnosis was confirmed by finding intermittently elevated urinary 17-hydroxysteroids, absent circadian rhythm, and elevated plasma ACTH. Plasma prolactin was slightly elevated. Films of the sella turcica were normal. A 9 mm basophilic microadenoma was removed by the transphenoidal approach. Immunocytochemical and electron-microscopic studies showed that the tumor was composed exclusively of ACTH secreting cells. Endocrine re-evaluation one year later revealed normal adrenal function. Serum prolactin had returned to normal. This case provides further evidence that Cushing's disease can be caused by a pituitary microadenoma insufficient in size to deform the sella.


Enzymatic and hemolytic properties of Propionibacterium acnes and related bacteria. The production of chondroitin sulfatase, hyaluronidase, deoxyribonuclease, gelatinase, phosphatase, lecithinase, and hemolysins was examined in 95 strains of Propionibacterium acnes and four related species of anaerobic, respectively, microaerophilic coryneform bacteria (P. avidum, P. lymphophilum, P. granulosum, and Corynebacterium minutissimum). All enzymes could be demonstrated in at least one representative of the species tested. Those Propionibacterium species most frequently found in acne vulgaris lesions, i.e., P. acnes and P. granulosum, proved to be the most active organisms concerning the production of the enzymes tested. P. avidum, on the other hand, showed the highest rate of hemolytic activity.


Use of immunoperoxidase for rapid diagnosis of mucocutaneous herpes simplex virus infection. The indirect immunoperoxidase method was applied to the rapid diagnosis of mucocutaneous herpes simplex virus infection. Specimens were obtained directly from lesions at various stages in their evolution. The immunoperoxidase method identified herpes simplex virus-infected cells in 91% of vesicular lesions which were positive by standard virological culture. At later stages, the number of cases that could be diagnosed decreased appreciably. The immunoperoxidase method appears to be a rapid and reliable method for the diagnosis of early herpes simplex virus infection.


Rotavirus isolation and cultivation in the presence of trypsin. Rotaviruses are generally difficult to isolate and culture in vitro; therefore, virus isolation has not been used as a method of diagnosing this group of agents. The present report describes a simple procedure for isolating bovine rotaviruses directly from feces after pretreatment of fecal samples with trypsin. This procedure resulted in virus isolation from five of five samples that contained virus particles, as demonstrated by electron microscopy, and four of seven samples where virus particles could not be observed but were considered positive by the presence of immunofluorescent-staining cells in feces. Virus could not be isolated from "normal" feces. If the virus was not passaged in the presence of trypsin, the infectivity was gradually lost, but infectivity could be restored again if trypsin was added, resulting in increased virus spread and concomitant increase in virus yield. The application of this technique as a diagnostic tool for bovine and other rotaviruses is briefly discussed.


Anti-complement immunofluorescence test for antibodies to human cytomegalovirus. An anti-complement immunofluorescence (ACIF) test that detects human cytomegalovirus (CMV) antigen in the nuclei of infected cells was used for assay of CMV antibodies in human sera. Various factors influencing the sensitivity and specificity of the ACIF test system were investigated, and results were applied to the development of a procedure which could be completed in a relatively short length of time and gave reproducible results. Results obtained in the ACIF test were compared with those obtained in complement fixation, indirect hemagglutination, and neutralization tests, and the ACIF test was shown to be suitable for detection of significant antibody titer rises and stationary levels of CMV antibody. Heterotypic antibody responses were not seen with sera from other human herpesvirus infections. The nonspecific cytoplasmic staining that occurs in indirect immunofluorescence tests for CMV did not occur in the ACIF system, and sera that were anti-complementary in complement fixation tests could be examined satisfactorily by ACIF. Thus, the test is a valuable supplemental or back-up procedure for the serodiagnosis of CMV infection.


Evaluation of anti-complement immunofluorescence test in cytomegalovirus infection. The anti-complement immunofluorescence (ACIF) technique was evaluated for the diagnosis of human cytomegalovirus (CMV) infection in a group of sera derived from renal transplant recipients and donors by comparing it with the indirect immunofluorescence (FA) and complement fixation (CF) TESTS. The ACIF and FA tests yielded similar results. However, the ACIF test had a distinct advantage over the indirect FA test, since it eliminated the nonspecific cytoplasmic staining that may result in false positive readings in inexperienced hands. Both the indirect FA and ACIF tests were more sensitive than the CF test. In primary CMV infection, the FA and ACIF antibodies appeared earlier and had significantly higher titer than corresponding CF titers. This difference in titers was not seen in seropositive individuals who lacked overt infection. Our previously reported correlation between the seropositivity of the donor and CMV infection in seronegative recipients has been confirmed.


Improved glycine-extracted complement-fixing antigen for human cytomegalovirus. High-titered, sensitive, and stable complement-fixing antigens for human cytomegalovirus were consistently produced from human diploid fibroblast cells infected at a high multiplicity of virus, harvested after 7 days of incubation, and sonically treated immediately in 0.1 M glycine buffer, pH 9.5.


Mechanisms contributing to malignant dysrhythmias induced by ischemia in the cat. Continuously recorded bipolar electrograms were obtained simultaneously from epi-, endo-, and mid-myocardial regions of the ischemic and normal zones of cat left ventricle in vivo after coronary occlusion, analyzed by computer, and compared to regional cyclic AMP levels. Regional cyclic AMP content was used as an index of the combined local effects of: (a) efferent sympathetic nerve discharge; (b) release of myocardial catecholamines due to ischemia; and (c) circulating catecholamines. Ischemia resulted in a progressive increase in pulse width and rise time and a decrease in rate of rise of voltage (dV/dt) of the local electrograms from ischemic zones reaching a maximum within 2.4+/-0.3 min (mean+/-SE) at the time of onset of severe ventricular dysrhythmias, all of which returned toward control before the cessation of the dysrhythmia (33.5+/-1.5 min after coronary occlusion). Increases in cyclic AMP in ischemic zones preceded corresponding increases in the frequency of premature ventricular complexes (PVCs). Propranolol inhibited the increases in cyclic AMP and reduced the frequency of PVCs in animals without ventricular fibrillation. In animals with ventricular fibrillation, cyclic AMP was significantly elevated in normal and ischemic zones compared to animals with PVCs only. Electrical induction of PVCs or ventricular fibrillation in ischemic and nonischemic hearts failed to increase cyclic AMP. The results suggest that the changes in regional adrenergic stimulation of the heart may contribute to perpetuation of ventricular dysrhythmia and the genesis of ventricular fibrillation early after the onset of myocardial ischemia.


Properties of the plasma very low and low density lipoproteins in Tangier disease. The absence of normal high density lipoproteins (HDL) in Tangier disease is well established, but the properties of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in this disorder have not been well defined. The profiles obtained by analytic ultracentrifugation and the chemical composition, morphology, and electrophoretic mobility of Tangier and normal VLDL and LDL were compared. Apolipoproteins were fractionated by gel chromatography and characterized by amino acid analysis, polyacrylamide-gel electrophoresis, and immunochemical reactivity. Concentrations of low density lipoproteins of S(f) (o) 0-12 were reduced in three of six Tangier plasmas studied by analytic ultracentrifugation. Accumulation of intermediate density lipoproteins (S(f) (o) 12-20) was not observed. Two subjects with hypertriglyceridemia had normal VLDL (S(f) (o) 20-400) levels, suggesting that abnormalities of chylomicron metabolism probably account for the hypertriglyceridemia frequently observed in this disorder. Tangier VLDL migrate more slowly than normal VLDL on paper electrophoresis, yet their morphology, gross chemical composition, and qualitative apolipoprotein content are similar. Quantitative abnormalities in C-apolipoproteins, however, were observed in Tangier VLDL. When patients were consuming unrestricted diets, C apoproteins accounted for 19-49% of the protein in lipoproteins of d < 1.006 g/ml. Ingestion of low-fat, high-carbohydrate diets reduced the VLDL-C-apoprotein content in all Tangier patients (mean = 17% of VLDL protein vs. 43% in controls). These findings suggested that a major proportion of the C apoproteins in Tangier plasma is associated with chylomicrons or their remnants, perhaps because the C-apoprotein reservoir normally provided by HDL is absent. This secondary mechanism for C-apoprotein conservation is lost when dietary fat is withdrawn.LDL-2 (1.035 < d < 1.063) from Tangier and control plasma had identical electrophoretic mobilities. Tangier LDL-2 had slightly smaller median diameters (210-225 A vs. 230-240 A in controls) and a quite different composition than normal LDL-2. Triglyceride accounted for a mean of 29% of Tangier LDL-2 mass (control = 6%) and the cholesteryl ester content was reduced by about 50%. Thus, HDL may be required for the generation of chemically normal LDL. Alternatively, the fundamental defect in Tangier disease may involve all lipoprotein classes.


Biologically active low density lipoprotein in human peripheral lymph. We have compared the ability of human serum and peripheral lymph to suppress the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), to activate cholesteryl ester synthesis, and to compete with 125I-labeled low density lipoprotein (LDL) for binding to LDL receptors in cultured human fibroblasts. Whole lymph was active in all three tests and the activity per unit volume in lymph was approximately equal to 1/10th that in serum. All three biologic activities in lymph were confined to the d less than 1.063 g/ml fraction. Whole lymph had no significant effect on HMG-CoA reductase activity in fibroblasts from a patient with homozygous familial hypercholesterolemia, whose cells lack LDL receptors. The LDL-like biologic activity per unit mass of immunologically active apoprotein B was approximately the same in lymph as in serum. The current data indicate that functionally active LDL is present in lymph and that the concentration of this lipoprotein is approximately equal to 1/10th that in serum.


Astrovirus associated gastroenteritis in a children's ward. During an outbreak of gastroenteritis in a paediatric ward astroviruses were found in faeces from 17 to 27 symptomatic children and from four of 14 members of the staff with diarrhoea. No viruses were found in 10 asymptomatic children. Fourteen of the 21 astrovirus excretors were free of any recognised pathogens, but in the other seven, rotaviruses or pathogenic bacteria were also present. Serological evidence of astrovirus infection was obtained in five adults and two children.


Prolyl hydroxylase in the skin of patients with obstructive jaundice. Prolyl hydroxylase was estimated in the skin of 24 jaundiced patients and 25 non-jaundiced patients. The activity of this enzyme was markedly reduced in the skin of the jaundiced patients.


Multiple myeloma and oat cell carcinoma. An association linking multiple myeloma with a higher incidence of carcinomatous tumours has been reported. The following case report describes a man diagnosed as having bronchogenic carcinoma found also to have myelomatosis. Aspects of this association are discussed.


Insulin stimulation of particulate, low Km adenosine 3',5'-monophosphate phosphodiesterase activity in fat cells of the obese-hyperglycemic (ob/ob) mouse. Cyclic AMP phosphodiesterase activity was examined in particulate (30,000 X g for 30 min sediment) and supernatant subcellular fractions of epididymal fat cells isolated from obese-hyperglycemic (ob/ob) mice and their lean (+/?) LITTERMATES. The activity of the enzyme(s) was measured during both the early onset phase (5-6 weeks of age) and the static (5 months of age) of the obese-hyperglycemia syndrome. Fat cell particulate and supernate cyclic AMP phosphodiesterase activity of obese-hyperglycemic mice and their lean littermates at both ages displayed nonlinear Lineweaver-Burk kinetic plots. The maximum velocities of the fat cell particulate cyclic AMP phosphodiesterase activity of the obese mice were 67% and 84% lower than those of their lean littermates at 5-6 weeks and 5 months of age, respectively. Incubating fat cells obtained from either lean or obese mice of both age groups with 30 to 240 microunits of insulin per ml for 15 min increased the activity of the particulate, low Km cyclic AMP phosphodiesterase. This increase in activity was manifest as an increase in the maximum velocity of the enzyme(s) with no significant alteration of the affinity of the enzyme(s) for cyclic AMP.


Properties of adenylate cyclase solubilized from rat adrenal membranes: effects of ACTH and other stimulators on solubilization. We have solubilized adenylate cyclase in a relatively stable form from rat adrenal membranes. The solubilized enzyme elutes on a column of Sepharose 4BR as a distinct peak with a higher molecular weight than the soluble fractions which bind 125I-ACTH. Both the soluble and membrane bound enzymes are activated by NaF and Gpp(NH)p, and both have similar affinities for MgATP. While the membrane bound enzyme is activated similarly by either Mg2+ or Mn2+, the soluble enzyme is more fully activated by Mn2+. Pretreatment of adrenal membranes with NaF or Gpp(NH)p before the addition of detergent enhances recovery of soluble enzyme activity, while recovery of activity in the unsolubilized membrane pellet is unchanged. In contrast, addition of ACTH prevents solubilization of the enzyme and greatly increases its recovery in the pellet. This observation is consistent with the theory that action of the hormone on a receptor subunit leads to an association between the receptor and a catalytic subunit. Such an association might make it more difficult to remove the enzyme from the surrounding lipid matrix of the membrane.


Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells. Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.


Effects of GTP on cyclic AMP concentrations in intact Ehrlich ascites tumor cells. Experiments were conducted to test the hypothesis that intracellular concentrations of guanosine triphosphate (GTP) regulate the activity and hormonal sensitivity of adenylate cyclase in intact cells. By appropriate treatments, the GTP concentrations of Ehrlich ascites tumor cells could be varied between 28% and 680% of control values. Cyclic AMP concentrations were measured before and after addition of epinephrine in cells containing this range of GTP concentratins. Basal cyclic AMP concentrations were unaffected by changes in GTP concentrations. In cells containing lowered concentrations of GTP, the cyclic AMP concentration following addition of epinephrine was half that in control cells. Elevation of GTP concentrations above normal and had no effect on cyclic AMP concentrations following epinephrine treatment.


Elevation of the cyclic GMP concentration in human platelets by sodium ascorbate and 5-hydroxytryptamine. Sodium L-ascorbate (ascorbate) and sodium D-ascorbate produced a dose-related rise of guanosine 3':5'-cyclic monophosphate (cGMP) in platelets with a maximum increment averaging 25-fold at 5 mM ascorbate. The ascorbate-induced increment in cGMP reached a peak after 1 min and was maintained for 1 h in the presence of ascorbate. 5-hydroxytryptamine (5-HT) also produced a dose-related rise of cGMP in platelets with a peak effect of approximately 25-fold at 16 micrometer 5-HT. The elevation of cGMP in platelets by both ascorbate and 5-HT did not require extracellular calcium and was blocked by inhibitors of cyclo-oxygenase such as aspirin or indomethacin. A maximum ascorbate-induced rise in platelet cGMP at the time of addition of epinephrine, collage or thrombin did not augment the release of [14C]5-hydroxytryptamine ([14C]5-HT) measured over 30 min. Although ascorbate appeared to increase platelet cGMP by modulation of endoperoxide formation, its failure to aggregate platelets or to influence the release reaction indicates that the ascorbate-stimulated rise in cGMP does not have a simple relationship to thromboxane formation.


Regional levels of cyclic AMP in rat brain: pitfalls of microwave inactivation. Techniques of in-vivo microwave irradiation to inactivate brain enzymes in rats were varied as to exposure configuration and output power. The rate at which metabolism was stopped was studied in various regions of the rat brain, using changes in levels of cyclic AMP and phosphodiesterase activity. Exposure times required to obtain stabilized levels of cyclic AMP varied in different brain regions, i.e., hypothalamus, cortex and cerebellum. Levels of cyclic AMP in selective regions of the brain decreased as more rapid inactivation was achieved. The authors identify important sources of variability of present microwave inactivation systems and the need for improved control of signficant microwave parameters.


Dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans. Dermatofibrosarcoma protuberans is a well-differentiated sarcoma that rarely metastasizes but is characterized by a high recurrence rate. This is due to clinically undetectable, microscopic extensions into the surrounding dermis. Chemosurgery (Mohs) is effective in the ablation of this tumor because the extensions can be identified and eradicated.


Comparative psychologic effects of estrogen administration on institutional and noninstitutional elderly women. Previous research on elderly women living in institutions has showm a postiive relationship between the administration of estrogen and improved psychologic status. In the current 12-month study, the effects of estrogen therapy were compared in 18 institutional women versus 21 noninstitutional women who volunteered to participate. Both groups received conjugated estrogens (Premarin, 0.3 mg daily) in interrupted courses. Throughout the year, the women were evaluated by means of the Eysenck Personality Inventory, the Profile of Mood States, the Lawson Morale Scale, and various self-perception items. Statistically, significant changes occurred in the areas of neuroticism, tension, morale, and subjective mood states, but the improvement was greater in the institutional subjects. The main conclusion was that estrogen therapy has a positive effect upon psychologic status, particularly for women who reside in an institution.


Two-step separation of human peripheral blood monocytes on discontinuous density gradients of colloidal silica-polyvinylpyrrolidinone. Normal human peripheral blood monocytes were purified by a two-step separation. The first step, the standard Ficoll--Hypaque (F--H) buoyant density centrifugation, yielded mainly mononuclear cells, of which 24 +/- 9% were monocytes. Isopycnic centrifugation on discontinuous gradients of colloidal silica polyvinylpyrrolidinone (CS-PVP) further separated these mononuclear cells. The density interface between 1.070 and 1.060 g/ml yielded 82 +/- 7% monocytes, 5 +/- 4% granulocytes and 13 +/- 8% lymphocytes. Sixty-six percent of the monocytes obtained after F--H separation were recovered in this layer. The monocytes were intact and viable and retained their ability to phagocytose and kill Candida pseudotropicalis and to spread on glass coverslips. Motility (both random and towards a chemoattractant) was retained but was quantitatively less than after F--H separation alone. The relative purity of the monocyte population allowed assessment of major histocompatibility surface antigens by serotyping. This confirmed the presence of HLA and Ia-like antigens on monocytes.


Oxidative phosphorylation in rat oral mucosal mitochondria. Oral mucosal mitochondria were isolated and characterized morphologically by electron microscopy. Polarographic measurements were made of respiration and oxidative phosphorylation in the mitochondrial preparations. ADP:O ratios approaching or slightly exceeding the theoretical maxima and stabilized respiratory control ratios were achieved with malate + glutamate, succinate and ascorbate-N,N,N1N1 tetramethyl-p-phenylenediamine (TMPD) as substrates. Inhibition by rotenone, antimycin A, azide, and cyanide established the classical electron transport chain as the major pathway of mitochondrial respiration. Respiration of the oral mucosal mitochondria was stimulated by DNP in the presence of succinate. DNP-stimulated respiration exceeded that observed in the presence of ADP plus Pi and increasing the concentration of DNP progressively inhibited respiration.


Activation of the kallikrein system in hyperbetalipoproteinemia. In 30 patients with hyperlipoproteinemia (19 type II and 11 type IV), the role of the intrinsic coagulation pathway was evaluated by assays of preK, Kl's, and Hageman factor (factor XII). Analysis of the plasma of type II patients suggested activation of the intrinsic pathway characterized by a 40% decrease in preK (p less than 0.01) and a 50% decrease Kl (p less than 0.01); in contrast, analysis of the plasma of type IV patients showed no activation of the intrinsic pathway. In type II patients C-1INH was present in normal concentrations as measured by immunochemical techniques. The findings of normal levels of C-1INH by immunoassay, in association with altered electrophoretic mobility, are suggestive of formation of a kallikrein-C-1INH complex in vivo.


Some ultrastructural features of acinic cell carcinoma. The ultrastructure of an acinic cell carcinoma, occurring in the left parotid gland of a 52-year-old woman and causing a total facial nerve paralysis, is described. Histologically the tumour consisted of numerous granulated cells arranged around lumen-like openings and resembling a secretory system. Furthermore, areas with agranulated cells growing in a solid pattern were also encountered. In the electron microscope the cytoplasmic granules of the tumour cells displayed a varied appearance. Granules of a dense homogeneous type, as well as granules with a more electron lucid appearance were observed. Furthermore, numerous cytoplasmic granules displayed a bipartite structure with a dense central and a more electron lucid outer zone. In specimens primarily fixed in OSO4 or KMnO4 the granules displayed a 'leached out' appearance. The membrane-bounded of the tumour cells also showed a strong positive staining with the periodic acid-chromic silver technique of Rambourg et al. (1969). Other characteristic ultrastructural features of the tumour cells studied were: Smooth cell surfaces, the presence of subplasmalemmal bands of electron dense material, desmosome-like attachment areas between cells and grossly altered mitochondria.


Action of anthocyanosides of Vaccinium myrtillis on the permeability of the blood brain barrier. Proteases and especially collagenase injected into the lateral brain ventricles of rats are able to increase the permeability of the blood-brain barrier to trypan blue. Treatment of the rats with anthocyanosides of Vaccinium myrtillis diminishes the permeability increasing effect of collagenase and accelerate the recovery of normal permeability. This effect seems to be related to a less effective enzymatic attack on collagen, as hydroxyproline content in the CSF is increased less after collagenase injection in treated animals than in untreated controls.


Autoradiographic localization of the binding of 125I-labelled prolactin to rat tissues in vitro. The binding of 125I-labelled prolactin to normal rat tissues was examined with autoradiographic techniques. Tissue slices were incubated with 125I-labelled ovine prolactin in the presence or absence of excess unlabelled hormone to distinguish between the specific and the non-specific localization of grains. Specific binding was found in the liver, adrenal gland and kidney from female rats, and in the mammary gland, ovary, testis and prostate gland. Conversely, fat, muscle, heart, lung, and spleen from female rats and uterine tissue did not bind 125I-labelled prolactin appreciably, or show competition for binding in the presence of unlabelled hormone. In the testis, prolactin bound exclusively to Leydig cells; in the prostate gland, binding was localized in the secretory epithelium. Kidney tubules in the cortex displayed specific prolactin localization whereas the medulla did not bind prolactin. Adrenal medulla showed no hormone binding; however, the zona reticularis and to a lesser extent the zona fasciculata bound prolactin. In the ovary, grains were limited to the theca and to a lesser extent the corpus luteum and follicles. In all cases, binding was essentially abolished when unlabelled prolactin was included in the incubation medium. These results confirm reports of the presence of prolactin receptors in these tissues and serve to identify the cells within a particular organ that respond to prolactin.


Metabolic effects of cortisol, corticosterone and adrenocorticotrophin in a prototherian mammal Tachyglossus aculeatus (Shaw). The effects of injections of cortisol, corticosterone and ACTH on indices of carbohydrate, fat and protein metabolism were investigated in the conscious echidna, Tachyglossus aculeatus. Intravenous infusion of cortisol and corticosterone for 2 h at rates of 3 and 30 microgram/kg/h respectively did not cause significant changes in the plasma concentrations of glucose, urea or amino acids during a 12.5 h observation period. In contrast, a dose-related increase in plasma free fatty acid (FFA) concentration was observed. Infusion of synthetic ACTH at 2 i.u./kg/h for 2 h caused a minor, short-lived increase in FFA concentration. Daily i.m. injections of 0.2 mg cortisol or corticosterone acetates/kg, which raised plasma total corticosteroid concentrations to levels characteristic of maximal ACTH stimulation, did not cause glycosuria nor was there any change in body weight, nitrogen intake or urinary nitrogen excretion. However, there was a minor, but significant, increase in plasma glucose concentration. The liver glycogen content of 24 h fasted, corticosteroid-treated animals was similar to that of fasted control animals. It is concluded that cortisol, corticosterone and ACTH have only minor effects on carbohydrate and protein metabolism and that the main action of these hormones may be to mobilize fat reserves.


Stimulation of prostaglandin accumulation in the rat anterior pituitary gland by luteinizing hormone releasing hormone in vitro. The addition of luteinizing hormone releasing hormone releasing hormone (LH-RH) to cultures of monolayers of rat anterior pituitary cells was shown to increase both the concentrations of prostaglandins E1 and E2 (PGE) in the cells and the release of LH over similar ranges of concentrations of LH-RH (10(-6) to 10(-10) mol/l). The peak concentration of PGE was observed after 2.5 h. The stimulation of the level of PGE in the cells by LH-RH was completely inhibited by two inhibitors of prostaglandin synthetase, which only partially inhibited the stimulation of LH release. Therefore the increased concentration of PGE was not obligatory for the effect of LH-RH on LH release. It was also shown that monobutyryl cyclic AMP stimulated the intracellular concentration of PGE and it is suggested that the stimulation of PGE levels may be mediated by increased levels of cyclic AMP in the cells after the addition of LH-RH.


Distribution of corticotrophin releasing factor activity within the hypothalamic-pituitary complex of rats and cattle. An assay system involving cultured rat adenohypophysial cells from either intact or adrenalectomized donors was used to study the distribution of corticotrophin releasing factor (CRF) activity in the hypothalamic-pituitary complex of rats and cattle. In the rat hypothalamus, CRF activity was most concentrated in the median eminence, but CRF was present in the stalk and the posterior pituitary gland in much higher concentrations than in the median eminence in both species. The dose--response slopes for the median eminence, stalk and pars nervosa of the posterior pituitary gland were parallel to each other, suggesting a qualitative similarity between the CRF activity in these tissues. Rat posterior pituitary glands may also contain another CRF component which has a much flatter dose--response curve, but is detectable in smaller quantities of posterior pituitary tissue than is the other type of CRF.


Interaction of thyrotrophin with the human thyrotrophin receptor. The relationship between the binding of thyrotrophin (TSH) to receptors and the production of cyclic AMP has been investigated using crude human thyroid membranes. Receptor binding was studied by use of 125I-labelled bovine TSH, which had been purified by adsorption to and elution from human thyroid membranes. This 125I-labelled material showed the same biological activity as the unlabelled hormone. Binding studies at equilibrium indicated that the association constant of the TSH-membrane interaction decreased as the amount of TSH bound increased. Kinetic data did not provide definite evidence that this was due to an effect of increasing receptor occupancy on the dissociation rate constant. Detectable stimulation by TSH of cyclic AMP production by the membranes was observed with as little as 30 micromicron. (1 ng) hormone. Increasing amounts of TSH over the range 0-250 micromicron caused increases in the production of cyclic AMP, proportional to the amount of hormone bound. With larger amounts of TSH, increasingly greater amounts of bound hormone were required to give corresponding increases in cyclic AMP formation, and addition of TSH in amounts greater than 16 millimicron resulted in progressive inhibition of cyclic AMP formation. Kinetic studies indicated that receptor binding was not rate-limiting in the stimulation of cyclic AMP production by TSH.


Interaction of thyroid-stimulating antibodies with the human thyrotrophin receptor. Thyroid-stimulating antibodies (TSAb) were found to inhibit the binding of labelled thyrotrophin (TSH) to thyroid membranes in a dose-dependent manner and this effect was localized in the Fab part of the TSAb molecule. Analysis of the binding data suggested that TSAb and TSH bound to the same receptor site. Production of cyclic AMP by the thyroid membranes was stimulated by TSAb and TSAb-Fab with a similar time course to that observed with TSH. Kinetic studies indicated that the binding of TSAb to the thyroid membranes was not rate-limiting in the process of stimulation of cyclic AMP production.


Effects of low-chloride solutions on action potentials of sheep cardiac Purkinje fibers. The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode's solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode's solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode's solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.


Electrical and adaptive properties of rod photoreceptors in Bufo marinus. II. Effects of cyclic nucleotides and prostaglandins. Substances known to alter cyclic nucleotide levels in cells were applied to the isolated toad retina and effects on rod electrical and adaptive behavior were studied. The retina was continually superfused in control ringer's or ringer's containing one or a combination of drugs, and rod activity was recorded intracellularly. Superfusion with cGMP, Bu(2)GMP, isobutylmethylxanthine (IBMX; a phosphodiesterase inhibitor), or PGF(2alpha) (a prostaglandin) caused effects in rods that closely match those observed when extracellular Ca(2+) levels were lowered. For example, short exposures (up to 6 min) of the retina to these substances caused depolarization of the membrane potential, increase in response amplitudes, and some changes in waveform; but under dark-adapted or partially light-adapted conditions receptor sensitivity was virtually unaffected. That is, the position of the V-log I curve on the intensity axis was determined by the prevailing light level, not by drug level. These drugs, like lowered extracellular Ca(2+), also decreased the period of receptor saturation after a bright-adapting flash, resulting in an acceleration of the onset of membrane and sensitivity recovery during dark adaptation. Long-term (6-15 min) exposure of a dark-adapted retina to 5 mM IBMX or a combination of IBMX and cGMP caused a loss of response amplitude and a desensitization of the rods that was similar to that observed in rods after a long-term low Ca(2+) (10(-9)M) treatment. Application of high (3.2 mM) Ca(2+) to the retina blocked the effects of applied Bu(2)cGMP. PGE(1) superfusion mimicked the effects of increasing extracellular Ca(2+). The results show that increased cGMP and lowered Ca(2+) produce similar alterations in the electrical activity of rods. These findings suggest that Ca(2+) and cGMP are interrelated messengers. We speculate that low Ca(2+) may lead to increased intracellular cGMP, and/or that applied cGMP, and/or that applied cGMP may lower cytosol Ca(2+), perhaps by stimulating Ca(2+)- ATPase pumps in the outer segment.


Regulation of synthesis of benzyl alcohol dehydrogenase in Acinetobacter calcoaceticus NCIB8250. Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0.2 h-1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of L(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carbon-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by L(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second type of repression was observed during rapid growth or after the addition of compound such as succinate which are rapidly and completely metabolized.


Increased numbers of heat-resistnat spores produced by two strains of Clostridium perfringens bearing temperate phage s9. Sporulation kinetics and spore heat resistance data were compared for a lysogenic strain of Clostridium perfringens, s9, before and after curing with ultraviolet irradiation. The cured strain showed the same growth rate in broth media as the lysogenic strain but took 6 h longer to form refractile spores. For lysogenized and cured strains the percentages of refractile spores produced that were heat-resistant (80 degrees C for 15 min) were 50 and 0.2, respectively. When reinfected with the temperature phage, the cured strain produced spores in 2 to 3 h, like the original lysogenic culture, and 10% of the spores produced were heat-resistnat.


Psychiatric follow-up studies. Practical procedures and ethical concerns. The difficulties of performing prospective psychiatric follow-up studies have recently increased due to growing concern over the ethical issues involved. In a study to determine the efficacy of different lengths of psychiatric hospitalization 235 patients were followed for 2 years after study hospitalization. Data were obtained on more than 90 per cent of the patients. The cost of performing 100 follow-up interviews ranged from $9,169 for patients living within a 6-mile radius of the study site, to $17,942 for patients living within the contiguous United States. It is concluded that through persistent effort useful data can be obtained by follow-up of psychiatric patients, while maintaining ethical standards which include respect for the rights and wishes of the patient.


Therapeutic embolisation of the external carotid arterial tree. Intra-arterial embolisation is a valuable adjunct to the treatment of many vascular lesions, including neoplasms such as glomus tumours or juvenile angiofibromas, and arteriovenous malformations. Its place in the management of the individual patient should be established before any surgical procedure is carried out, as this may prejudice the eventual result. The indications for the procedure, the technique, and possible complications are discussed in this paper, and it is emphasised that the latter are best avoided by the use of a scrupulous technique and adequate technical facilities.


Median--ulnar nerve communications and carpal tunnel syndrome. Carpal tunnel syndrome in the presence of anomalous median to ulnar nerve communications in the forearm produces a characteristic change in motor conduction studies. Median nerve stimulation at the elbow evokes a thenar muscle action potential (MAP) with an initial positive deflection not seen on stimulation at the wrist. In 63 patients this change occurred in 16 (25%) and is a useful additional criterion in the diagnosis of carpal tunnel syndrome. The initial positive deflection is due to the volume-conducted MAP from the first dorsal interosseous and some thenar muscles whose motor point lies some distance from the recording electrode over abductor pollicis brevis. The first dorsal interosseous and thenar MAPs resulting from elbow stimulation of those median nerve axons crossing to ulnar nerve in forearm, are generated before that from thenar muscles supplied by the axons going through the carpal tunnel.


Sensory conduction in medial plantar nerve: normal values, clinical applications, and a comparison with the sural and upper limb sensory nerve action potentials in peripheral neuropathy. A method for recording the medial plantar sensory nerve action potential at the ankle with surface electrodes is described. Normal values in 69 control subjects are given and compared with the sural sensory nerve action potential in the same limb in the same subjects. Clinical applications were studied in 33 patients. The procedure may be applied in the diagnosis of L4-5 nerve plexus or root lesions, lesions of the sciatic, posterior tibial, and medial plantar nerves, and is a more sensitive test than other sensory nerve action potentials in the diagnosis of peripheral neuropathy.


Latent herpes simplex virus infection of mice. Infectious virus in homogenates of latently infected dorsal root ganglia. C-57 albino weanling mice were latently infected with herpes simplex virus (Mp strain, type 1) by inoculation of 10(4) plaque forming units in the right hind footpad. The virus was demonstrable in explant cultures of the sacral dorsal root ganglia of these mice for as long as 18 months following inoculation. In addition, the virus was detectable when homogenates of these latently infected ganglia were placed on to differentiated organotypic cultures of fetal mouse dorsal root ganglia for as long as 8 months following inoculation of the mice. Virus was not demonstrable in these homogenates when they were placed on the Hela cells. The results suggest that during herpes simplex virus latent infection in mice there is continuous synthesis of infectious virus, probably in a highly localized area, which is detectable if a sensitive indicator substrate, such as these organotypic cultures, is used.


Changes of the neuronal endoplasmic reticulum in the peripheral nervous system in mutant hamsters with hind leg paralysis and normal controls. The fine structure of the spinal root and gasserian ganglia of the mutant hamster with hind leg paralysis and of normal controls was examined. Three types of whorl-like or lamellar alteration of the endoplasmic reticulum were encountered but these were also found in the normal control animals and are therefore probably not related to the paralysis of the mutants. These alterations of the endoplasmic reticulum were similar to or identical with those seen in a variety of other conditions and in other types of cells. In the hamster, however the three configurations could sometimes be seen within the same membranous accumulations and are probably related to one another in a developmental fashion.


Transsphenoidal microsurgical removal of 250 pituitary adenomas. In a series of 250 pituitary adenomas, 72 (28.8%) were nonsecreting and 178 (71.2%) produced a hypersecretion syndrome: human growth hormone (83), prolactin (59), and adrenocorticotropic hormone (ACTH) (36). One-fifth had received prior treatment and one-fourth had visual impairment. The technical aspects of the transsphenoidal procedure are given with separate consideration of microadenomas and larger tumors. The results are provided in summary form with emphasis on the favorable outcome following removal of microadenomas. There was one postoperative death, and the complications observed after operation are presented.


The ultrastructure of tubular carcinoma of the breast. The ultrastructure of four tubular carcinomas of the breast is described. The neoplastic tubules are single layered and lack a myo-epithelial component, an important feature in distinguishing tubular carcinoma from sclerosing adenosis. Numerous myo-fibroblasts are present in the stroma of all four tumours and, since cells of this type have been recorded only occasionally in the stroma of other types of breast carcinoma, it is suggested that they may be a characteristic feature of tubular carcinomas.


Cyclic AMP and the mechanism of leucocyte lysosomal enzyme release during an immediate hypersensitivity reaction in vivo. The pleural cavity of rats was used to study the effect of altering leucocyte cyclic AMP content on the release of B-glucuronidase activity during an immediate hypersensitivity reaction. The effect on intravenous colchicine was also studied. Despite an increase of 135 to 235% in leucocyte cyclic AMP content no decrease in B-glucuronidase release was observed. Similarly, colchicine had no effect on enzyme release. It was concluded that the cyclic nucleotides and leucocyte microtubules may have no significant role to play in the release of lysosomal enzymes during acute inflammation in vivo.


Tyrosinemia associated with perinatal infection with cytomegalovirus. A premature infant presented with elevated concentrations of tyrosine in blood and urine, evidence of hepatocellular damage, demineralization of the bones, and a renal Fanconi syndrome. This is the clinical picture found in hereditary tyrosinemia. The infant also had a perinatal infection with cytomegalovirus.


Preoperative irradiation and chemotherapy for initially unresectable hepatoblastoma. Three cases of hepatoblastoma that were deemed unresectable, both by angiographic study and exploration, became resectable following combined therapy of dactinomycin, vincristine, and irradiation.


Computed tomographic scanning in children. Computed tomographic scanning (CT) has been effectively utilized in evaluating the pediatric patient. Our experience with CT of the chest and abdomen in 19 patients has been described. The advantages offered by CT include: a unique anatomic display, the lack of morbidity, and an acceptable radiation dosage. Few technical disadvantages exist. Further application of this unique technique can be predicted.


The effect of pancuronium and gallamine on muscarinic transmission in the superior cervical ganglion. Pancuronium enhanced contraction of the nictitating membrane elicited via ganglionic muscarinic pathways in the superior cervical ganglion of the cat. In order to elucidate this phenomenon, recordings of the superior cervical ganglion surface potential were made which demonstrated that pancuronium and gallamine reduced and haloperidol enhanced ganglionic hyperpolarization without significantly altering the ganglionic slow depolarization. Pancuronium reversed the effects produced by haloperidol, whereas the latter drug was unable to antagonize those induced by pancuronium. These results allow the speculation that pharmacologically distinct muscarinic receptors reside in sympathetic ganglia, one of which is susceptible to blockade by pancuronium or gallamine.


Substance P and Renshaw cells: a new concept of inhibitory synaptic interactions. 1. The actions of microelectrophoretically administered substance P on Renshaw cells in pentobarbitone anaesthetized cats were investigated. 2. The effects on spontaneous and synaptic firing and interactions with a number of other agents including acetylcholine, acetyl-beta-methylcholine, acidic amino acids, morphine, dihydro-beta-erythroidine and strychnine were studied in attempts to elucidate the mechanism of action of substance P. 3. Substance P usually selectively depressed the excitation by ACh, and also reduced submaximal synaptically evoked discharges which activate nicotinic receptors, but failed to modify excitation caused either by acetyl-beta-methylcholine, which activates muscarinic receptors, or excitation caused by glutamate or homocysteate. Substance P also depressed excitation by morphine which acted via the nicotinic receptors. 4. The inhibitory effect was not blocked by strychinine and was considered to be unlikely to be due to interaction between the polypeptide and either glycine or GABA receptors. 5. On some cells substance P caused excitation which was blocked by dihydro-beta-erythroidine. Mixed excitatory-inhibitory effects were observed on some of these neurones. 6. The results are discussed in relation to the possibility that substance P could function as a synaptic inhibitory mediator with an unusual selectivity of action.


Calcium requirement for activation of intact aortic smooth muscle. 1. Elevation of [K(+)](e) induced a contraction of rabbit aorta. If 10 mM-La(3+) was applied to rabbit aortae prior to [K(+)](e) elevation no contraction occurred. When 10 mM-La(3+) was applied simultaneously with, or at short time periods after, elevation of [K(+)](e) graded contractions were obtained whose magnitudes were higher if La(3+) was added at a later stage.2. Net cellular Ca fluxes associated with these graded contractions were measured using (45)Ca as well as atomic absorption. Correlation of the graded contractions with the net inward Ca movements yielded a Ca activation curve for intact arterial smooth muscle cells. The curve was S-shaped with half-maximal activation at a net Ca uptake of 41 mumole/kg wet wt. of aorta and full activation at 64 mumole/kg. If corrections were made for the extracellular space, full activation occurred at a net Ca uptake of 102 mumole/kg smooth muscle cells or roughly 25 mumole in excess of the value calculated to bind to the activation sites on arterial smooth muscle myosin.3. The contractile force of the smooth muscle cells not only depended on the magnitude, but also on the rate of the net Ca uptake.4. Elevation of tissue c-AMP through inhibition of phosphodiesterase greatly reduced both the rate and magnitude of contraction without affecting the size of the high K(+) stimulated Ca influx. It was concluded that high K(+)-induced depolarization activates the rabbit aorta by stimulating a net Ca uptake, and that the degree of activation is modified by c-AMP sensitive Ca accumulation by intracellular organelles.


Quantitative structure-activity relationships in centrally acting imidazolidines structurally related to clonidine. The central hypotensive action of clonidine and 26 structurally related derivatives was quantified by means of an ED30 obtained from dose-response curves following intravenous administration to anesthetized, normotensive rats. Multiple regression analyses of the biological data yielded correlation equations comprising a relationship between hypotensive activity and molecular structure. In the equations the pharmacokinetics together with the actual engagement of the central alpha-adrenoceptor are accounted for. More detailed characteristics of this central alpha-adrenoceptor emerged from correlation studies in which new ED30 values, associated with brain concentrations, were employed. The use of this biological parameter at the alpha-adrenoceptor level allowed the presentation of a hypothetical working model for the mechanism of interaction between this receptive site and clonidine-like imidazolidines.


Molecular orbital studies on the mechanism of drug-receptor interaction. 2. beta-Adrenergic drugs. An approach to explain the role of the aromatic moiety. The role of the aromatic moiety of beta-adrenergic drugs in the interaction with the receptor was investigated using the quantum mechanical ab initio SCF-MO-LCAO method. The structure-activity relationship was essentially discussed by analyzing the electrostatic molecular potential of three compounds which constitute meaningful portions of isoproterenol, INPEA, and doberol, the first drug having a stimulating activity and the others a blocking one. The results obtained point out the different roles played in the drug-receptor interaction by the various regions of the drugs and they also show that the aromatic moiety influences both the affinity and the intrinsic activity of the drugs. Indeed, the spatial correspondence among zones with negative potentials, which are localized on the phenyl substitutents of isoproterenol and INPEA and on the phenyl ring of doberol, could contribute to the affinity. On the other hand, the intrinsic activity of isoproterenol might be associated both with the proton-donor tendency of one phenolic OH group and with the wide zone of negative potential which spreads on a large part of the aromati moiety.


1,2,4-Triazole amino nucleosides. 1-beta-D-3'-Amino-3'-deoxyribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides. The synthesis of 1-beta-D-3'-amino-3'-deoxyribofuranosyl-1,2,4-triazole-3-carboxamide--the 3'-amino analogue of ribavirin--and five related nucleoside analogues is described. Each analogue exhibited LD50 concentrations greater than 100 microgram/mL against P-388 mouse lymphoid leukemia cells in tissue culture. Antiviral testing indicated that none of the compounds exhibited significant activity.


Stimulation of hepatic microsomal metabolism in mice by a mixture of polybrominated biphenyls. The pattern of stimulation of hepatic microsomal mixed function oxidases has been studied in female NMRI mice following a single ip injection of 150 mg/kg polybrominated biphenyls (PBBs) and compared to the patterns produced by phenobarbital (PB), 3 X 100 mg/kg; 3-methylcholanthrene (MC), 3 X 20 mg/kg; and these two agents administered together at these doses. Cytochrome P450, ethylmorphine N-demethylase, and epoxide hydratase reached maximums of 200, 200, and 350% of control values, respectively, at 48 hr after treatment with PBBs. Ethoxycoumarin O-deethylase and arylhydrocarbon hydroxylase were maximally increased to 400 and 180% of control values, respectively, 96 hr after PBBs. The reduced cytochrome P450 ethylisocyanide difference spectra and the inhibition of ethoxycoumarin O-deethylase and arylhydrocarbon hydroxylase activity by metyrapone and alpha-naphthoflavone indicated that the characteristics of the cytochrome P450 and the cytochrome P450-dependent enzymes changed with time after administration of PBBs. These results indicate that the enzyme-stimulating properties of PBBs alter, changing from PB-like to MC-like, with time after administration. These findings provide an explanation for the effects of PBBs on the toxicity of bromobenzene, indicating that PBBs represent a new and previously unrecognized calss of toxicity-modifying agents sharing properties of both PB and MC.


Plasma osmolality and electrolytes during the post partum months in Ghanaian women. Plasma osmolality and electrolytes levels were determined in lactating women during the period of 7th week to the 18th month post partum and compared with the levels seen in a control group of non-pregnant non-lactating women of the same age group (18-30 years). On the whole, plasma sodium, chloride and osmolality levels in the lactating women were found to be slightly but significantly elevated, while the plasma potassium levels were lowered. We suggest that these are due to the effects of increased ACTH secretion that occurs during lactation.


Pulsatile tinnitus: the role of angiography. Pulse-synchronous tinnitus is probably due to turbulent blood flow in or around the temporal bone. The anatomy of temporal bone blood supply is reviewed. Eight examples of tinnitus are presented to illustrate the spectrum of lesions that will produce this symptom and the role of angiography in its investigation.


Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus. A fibroblast line of the 3T3 type with a low saturation density was established from Fisher rat embryo cells. After infection with either wild-type or tsa mutant polyoma virus, transformants were isolated and cloned at 33 degrees C on the basis of their ability either to grow as dense foci on plastic in liquid medium (type N) or to form colonies in soft agar (type A). Polyoma T antigen was detected in all of the transformed lines. The following growth characteristics were studied for both types at 33 and 41 degrees C: saturation density, growth in soft agar and at a low serum concentration, colony-forming ability, and generation time. tsa-N transformants behaved at 33 degrees C similarly to transformed cells, but reverted at 41 degrees C to the nontransformed phenotype for all of these characters. tsa-A transformants and all of the wild-type transformants exhibited the transformed phenotype at both low and high temperatures. These results led us to distinguish at least two types of virus-induced transformants. In one of them, the activity of the protein affected by the tsa mutation appears to be necessary for the expression of several of the characters defining the transformed state.


Genetic relatedness of equine herpesvirus types 1 and 3. The genetic relatedness of two types of equine herpesviruses (EHVs), 1 (EHV-1) and 3 (EHV-3), was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA probe was allowed to reassociate in the presence or absence of the second unlabeled viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating only 2 to 5% homology between the two EHV genomes. Moreover, labeled RNA extracted from EHV-3-infected cells hybridized to filter-immobilized EHV-1 DNA only 2 to 3 percent as efficiently as to the homologous EHV-3 DNA. These results demonstrate that the genital (EHV-3) and nongenital (EHV-1) types of EHVs exhibit very little genetic homology.


Slow switchover from host RNA synthesis to bacteriophage RNA synthesis after infection of Escherichia coli with a T4 mutant defective in the bacteriophage T4-induced unfolding of the host nucleoid. Most, if not all, host RNA synthesis was shut off after infection of Escherichia coli strain B/5 with a bacteriophage T4 multiple mutant defective in the abilities to induce (i) unfolding of the host nucleoid (unf-), (ii) nuclear disruption (ndd-), and (iii) host DNA degradation (denA-, denB-). The shutoff of host RNA synthesis and turn-on of phage RNA synthesis were slower after infection of E. coli with unf- phage than after infection with unf+ phage. This delay in the switchover from host RNA synthesis to phage RNA synthesis in unf- infections did not result in a measurable delay in the onset of nuclear disruption, deoxyribonucleoside monophosphate kinase synthesis, or DNA synthesis. unf39 did not complement alc (allows late transcription on cytosine-containing DNA) mutants, supporting the proposal of Sirotkin et al. [Nature (London) 265:28-32, 1977] that alc and unf are possibly the same gene.


Two methyltransferase activities in the purified virions of vesicular stomatitis virus. In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.


Polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus. Purified avian infectious bronchitis virus was digested with bromelain (0.7 mg/ml), and the surface projections were removed. Polyacrylamide gel electrophoresis of the polypeptides from these bromelain-treated particles showed that VP1, VP2, and VP5 were missing from the seven polypeptides. VP1 to VP7, that were present in untreated virus preparations. Milder bromelain treatment (0.07 mg/ml) left visible surface projections and polypeptides comprising VP1 and VP2 intact, but removed VP5. Thus, there are apparently two types of surface projections on the virus particle. The ribonucleoprotein complex was released from virus particles disrupted with 1% Nonidet P-40. The proportion of VP6 in such preparations was greatly reduced, implying that VP6 is the structural polypeptide of the ribonucleoprotein. Polypeptides VP1, VP2, VP4, and VP5 are glycosylated, but none of the polypeptides contains lipid.