Effects of testosterone on the behavior of male cynomolgus monkeys (Macaca fascicularis)

To determine the threshold doses of testosterone propionate (TP) that cause clear-cut behavioral changes in the sexual behavior of castrated male cynomolgus monkeys, observations were made on three males during successive 5-week treatment periods while they received daily subcutaneous doses of 100 μg TP increasing in octaves to 25.6 mg TP. Males were tested with each of the same two ovariectomized, estrogen-treated females (6 pairs, 330 1-hr behavior tests). To mimic the diurnal plasma testosterone rhythm, TP injections were given at 1600 hr and blood samples were obtained at 0800 hr (141 samples). Male ejaculatory activity increased at the threshold dose of 200 μg TP per day giving plasma testosterone levels of 830 ng/100 ml, which is in the physiological range of 600–1600 ng/100 ml for intact males. This threshold dose was eight times higher than in rhesus monkeys on a dose per kilogram body weight basis. There was a further marked increase in ejaculatory performance at higher doses (6.4 to 25.6 mg) giving supraphysiological plasma levels of 4000–9000 ng/100 ml. There were individual differences in the behavioral changes occurring with TP treatment, and the female partner modulated the effects. These findings were generally similar to those obtained with male rhesus monkeys, but certain species differences were noted.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

Acetylcholine receptor-controlled ion flux in electroplax membrane vesicles: A minimal mechanism based on rate measurements in the millisecond to minute time region

The dependence of acetylcholine receptor-controlled transmembrane ion flux on carbamylcholine concentration was measured in the msec time region, using membrane vesicles and a quench flow technique. 4 Measurements were made: (1) transmembrane ion influx, (2) rate of inactivation of the receptor by carbamylcholine, (3) rate of recovery, and (4) ion influx mediated by “inactivated” receptor. The minimal model, based on the measurements, accounts for the time dependence of receptor-controlled ion flux over a 200-fold carbamylcholine concentration range. The maximum flux rate of 84 sec^?1 indicates that we have succeeded in measuring the receptor-controlled processes which give rise to electrical signals in cells.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Equilibrium isoelectric focussing in acrylamide gel slabs--reduction of cathodic drift.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, ³²P-labeled RNA samples can be counted with better relative efficiencies and those labeled with ¹⁴C or ³³P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.     

The respiratory system of Pseudomonas putida: participation of cytochromes in electron transport

Rates of oxygen utilization by Pseudomonas putida respiratory particles were measured using the electron donors, reduced nicotinamide adenine dinucleotide (NADH) and succinate, and the oxidation-reduction dyes, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethyl-p-phenylenediamine. The maximal rates produced by NADH and succinate were similar for particles from either log- or stationary-phase cells, but rates measured using the dyes were much higher in stationary-phase particles. Cyanide and azide were very effective inhibitors of dye oxidation in both cases, but they produced only partial inhibition of NADH and succinate oxidation in log-phase particles and had no effect in the stationary phase. Spectral examination of the cytochromes at several levels of reduction produced by the various electron donors and inhibitors indicated that most of the cytochromes that were reduced by the dyes lie on a cyanide sensitive pathway of electron transport. These findings support the hypothesis that P. putida produces an electron transport system in the stationary phase which involves branching at the level of the cytochromes.Inhibition of oxygen utilization by CO was nearly complete for all four substrates in logphase particles. Inhibition was also reasonably effective for dye oxidation in the stationary phase, but there was no effect on NADH or succinate oxidation. Photochemical action spectra of the relief of CO inhibition revealed that NADH and succinate oxidation in log-phase particles probably involves cytochrome o. Oxidation of the dyes by either type of particles also appeared to involve cytochrome o, and the possibility of the participation of an a- or d-type cytochrome was also indicated.     

Effects of atractyloside and palmitoyl coenzyme A on calcium transport in cardiac mitochondria.

Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.     

Changes in rat renal cortex, isolated plasma membranes and urinary enzymes following the injection of mercuric chloride.

Some of the biochemical changes in rat kidney following the administration of mercuric chloride have been determined. Mercuric chloride had an immediate effect on the renal brush border resulting in rapid loss of the microvilli. Plasma membranes were isolated and characterised at various stages in the necrotic process, mircovilli were absent from these preparations and the activities of marker enzymes for the brush border were significantly decreased. In contrast the basal plasma membranes were unaffected by the nephrotoxin during the early stages and no change occurred in the activity of (Na+ + K+)-ATPase, a marker enzyme for the basal membranes. The change in the pattern of urinary enzyme excertion closely paralleled the ultrastructural changes in the tubular cells. The sequence of subcellular change following the administration of mercuric chloride is discussed in relation to the known mechanism of action of this agent.     

A new theory on the origin and the nature of viruses

The hypothetical model presented herein concerns the origin and nature of viruses. It advances the possibility of the appearance and existence of an organism lacking a cohesive morphological structure, that is: its subsystems are not in structural continuity. An attempt to delimit the concepts of life and organism and to integrate the viruses into this framework is made. Viruses are presented as organisms which pass in their ontogenetic cycle through two distinctive phenotypic phases: (1) the vegetative phase and (2) the phase of viral particle or nucleic acid. In the vegetative phase, considered herein to be the ontogenetically mature phase of viruses, their component molecules are dispersed within the host cell. In this phase the virus shows the major physiological properties of other organisms: metabolism, growth, and reproduction. Therefore, life is an effective presence. It is shown also, that in this phase so called "DNA viruses" have both nucleic acids: RNA as well as DNA. The virions are considered to be "spores" or reproductive forms of the virus, possessing life only as a potential property.     

Kinetic identification of component X as P430: a primary electron acceptor of Photosystem I

Kinetics of dark recoveries of Component X, Center A, and Center B at 20 and 0 °C after a 30-s illumination were studied in membrane fragments from a blue-green alga by using low temperature electron paramagnetic resonance spectroscopy in combination with a quick-freeze method. These kinetics were compared with those obtained by spectrophotometry under the same conditions. Contrary to the currently popular view, the result strongly suggests that Component X, rather than Center A or Center B, is P430.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase

The nucleotide sequence of 1400 bp from R-plasmid R538-1 containing the streptomycin/spectinomycin adenyltransferase gene (aadA) was determined, and the location of the aadA gene was identified by a combination of insertion and deletion mutants. Its gene product, aminoglycoside 3"-adenylyltransferase (AAD(3")(9), has a Mr of 31,600.     

Differences in the pattern of attack of acidic,neutral, and basic phospholipases A2 of A. halys blomhofii on human erythrocyte membranes: Problems in interpretation of phospholipid location

Purified acidic (pI 4.9), neutral (pI 6.9), and basic (pI 8.7) phospholipase A₂ from Agkistrodon halys blomhofii showed characteristically different patterns of hemolysis and phospholipid hydrolysis of intact human erthyrocytes. Acidic and neutral enzymes were nonlytic in the early periods of incubations with intact erythrocytes whereas the basic enzyme caused immediate hemolysis (5–8%). Under nonlytic conditions acidic and neutral enzymes hydrolyzed only phosphatidyl choline (PC) (20 and 50%, respectively), whereas basic enzyme hydrolyzed not only PC (60%) but nearly 15% of the phosphatidylethanolamine (PE). Both PC and PE were hydrolyzed significantly when the three phospholipases A₂ were incubated individually with erythrocyte lysate or hypotonic ghosts (sealed or unsealed). The order of substrate preference for acidic and neutral enzymes was always PC > PE. On the contrary basic enzyme exhibited the property of substrate specificity reversal. It hydrolyzed PC faster than PE when the membranes were sealed whereas PE hydrolysis was faster than PC hydrolysis in unsealed membranes. Interestingly only the basic enzyme showed activity in the absence of Ca²⁺ and in the presence of 0.5 mm EDTA. Phospholipase C (Bacillus cereus or Clostridium perfringens) did not show the property of substrate specificity reversal although their ability to hydrolyze PC and PE was different. In general this study demonstrates the unique activity patterns of three physically different pure phospholipases A₂ on human erythrocyte membranes which could be of value in selectively modifying membrane phospholipids. In addition it also throws an important light on the fact that results obtained with phospholipases should be interpreted with caution particularly as regards the localization of phospholipids in membranes.     

Lack of correlation between agglutinability,the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.