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.     

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.     

Environmental regulation of female calling and male pheromone response periodicities in the codling moth (Laspeyresia pomonella)

Codling moth female calling and male pheromone responsiveness under the defined conditions of 23°C and light:dark (LD) 16:8 occurred primarily during scotophase. Under either continuous photophase or scotophase females called with periodicities very similar to their periodicity under the LD cycle, indicating that the rhythmicity is circadian. Male response rhythmicity was maintained under continuous photophase. A decrease in the temperature from 23° to 16°C resulted in a reduction in the proportion calling when the decrease in temperature occurred during scotophase and a shift of maximal calling into photophase when the decrease in temperature occurred 3 hr prior to the initiation of scotophase. Decreases of temperature from 23° to 16°C and of light intensity did not produce similar shifts in the periodicity of male upwind orientation. Of 6 pheromone dosages from 10^?5 to 10² μg, 10^?1 and 10⁰ μg elicited the most male upwind orientation.     

Effects of chronically elevated intake of sweet solutions on sexual behavior of male rats

Three experiments were conducted on the sexual behavior of gonadally intact and castrated male Sabra rats. Half of the animals drank water during the course of the experiment and half were offered sweet solutions, the assumption being that sweet gustatory stimulation elevates the level of central endogenous opioid peptides in rats. The effects on sexual behavior of the following drugs were explored: the opiate receptor blocker naloxone (5 mg/kg, sc), the serotonin precursor 5-hydroxytryptophan (5-HTP) (20 mg/kg, sc), the serotonin antagonist methysergide (1 mg/kg, sc), and naloxone in combination with methysergide. Naloxone, whether administered alone or in combination with methysergide, impaired sexual performance in castrated male rats, and in gonadally intact rats maintained on sweet solutions. Methysergide elevated sexual behavior in all groups, whereas 5-HTP tended to suppress such behavior. The results support the hypothesis that endogenous opiates play a role in the expression of male sexual behavior in rats. While subtle in intact animals this role may become crucial following the disruption of sex hormone supply. Serotonergic influence on male sexual behavior is inhibitory.     

In vitro effect of various mitogens on starfish (Asterias rubens) axial organ cells

The effects of various mitogens on axial organ (AO) cells of the sea star have been studied. Pokeweed mitogen (PWM) stimulates [³H]thymidine incorporation by the whole population of axial organ cells of the sea star. This effect occurs 24 hr after the addition of PWM and is maximal at 40 μg/ml. In contrast, no stimulation is observed when coelomocytes are treated with PWM under the same conditions. No stimulation of the whole AO cell population is observed in the presence of Con A or LPS. However, the AO cell population can be divided, on the basis of surface adherence properties, into two subpopulations, adherent and nonadherent. Con A stimulates the nonadherent cells, but not the adherent cells: The stimulating effect is maximal 24 hr after addition of Con A and at 0.2–0.5 μg/ml. In contrast, LPS stimulates the adherent but not the nonadherent cells and the stimulating effect is maximal at 24 hr and at 45 μg/ml.     

Chemical investigations on the gum exudate from sajna (Moringa oleifera)

The purified, whole-gum exudate from the drum-stick plant (Moringa oleifera) was found to contain l-arabinose, d-galactose, d-glucuronic acid, l-rhamnose, d-mannose, and d-xylose in the molar ratios of ～ 14.5:11.3:3:2:1:1. A homogeneous, degraded-gum polysaccharide consisting of d-galactose, d-glucuronic acid, and d-mannose in the molar ratios of ～ 11.7:3.9:1, was obtained on mild hydrolysis of the whole gum with acid. Permethylation studies were conducted on the whole gum, the degraded gum, and their carboxyl-reduced products, and the results were in good agreement with those obtained from periodate oxidation followed by Smith degradation. Also, isolation and characterization of the oligosaccharides obtained from the mother liquor during preparation of the degraded gum, and by graded hydrolysis of the degraded gum, were achieved. On the basis of the results obtained from these studies, a tentative structure was assigned to the average repeating-unit of the gum.     

Effects of steroidal hormones on sexual, attack, and search behavior in the isolated male chick

Immature male chickens were treated with testosterone (1 mg/day), Δ₄-androstenedione (1 mg/day), 5α-dihydrotestosterone (5α-DHT; 1 mg/day), 5α-androstanedion (1 mg/day), or estradiol (100 μg/day) in order to assess the effects of these steroids on copulatory behavior, agonistic behavior, and attentional processes. Testosterone, estradiol, and 5α-DHT were most effective in stimulating male copulatory behavior above that of oil-treated controls; whereas Δ₄-androstenedione and 5α-androstanedione had less, but nevertheless significant, effects on this behavior. Testosterone and 5α-DHT facilitated agonistic behavior; however, estradiol, 5α-androstanedione, and Δ₄-androstenedione were ineffective in this capacity. The persistence of response to a given stimulus type was increased by testosterone and decreased by 5α-DHT: 5α-Androstanedione had no discernible effect on this behavior. These findings suggest that in the male chicken the neural structures regulating male copulatory and aggressive behavior as well as attentional processes are differentially sensitive to sex steroids. The effects of all these steroids on somatic structures were assessed.     

Mutagenicity and cytotoxicity of the carcinogen-mutagen aflatoxin B1 in Streptococcus pneumoniae (Pneumococcus) and Salmonella typhimurium: dependence on DNA repair functions

Strains R6, R6x and R6uvr-1 of Streptococcus pneumoniae (Pneumococcus) are sensitive to the cytotoxic effects of the mutagen/carcinogen aflatoxin B₁ (AFB₁). R6uvr-1 is more prone to the cytotoxic effects of AFB₁ than the repair-proficient parental strain, R6. The same differential susceptibility of strains R6, R6x and R6uvr-1 was observed when UV light replaced metabolically activated AFB₁. All pneumococcal strains were immutable by AFB₁. AFB₁ mutagenesis in Salmonella typhimurium strains was dependent on a functional RecA gene product. The enhancing effects of ΔuvrB and plasmid pKM101 were found to be additive. Data presented are consistent with the following: (i) AFB₁ toxic effects are due mainly to DNA binding of AFB₁; (ii) AFB₁ mutagenesis is dependent on error-prone DNA repair; (iii) Pneumococcus lacks an active error-prone (SOS) DNA-repair system.     

Structural investigations on two hemicellulosic polysaccharides from groundnut (Arachis hypogea) seed endosperm

A highly branched xylan and a linear, β-d-(1→4)-linked glucomannan are the two hemicellulosic components isolated from the endosperms of groundnut (Arachis hypogea). Electrophoretic, sedimentation, and sugar analysis indicate the polysaccharides to be fairly homogeneous. The O-methyl derivatives of the polysaccharides were analysed, after reduction and O-acetylation, by gas-liquid chromatography and g.l.c.-mass spectrometry. 2,3,4-Tri-O-methyl-d-xylose (3.6 mol), 2,3-di-O-methyl-d-xylose (21.0 mol), 3-O-methyl-d-xylose (2.8 mol), and d-xylose (4.2 mol) were detected in the xylan, whereas 2,3,4,6-tetra-O-methyl-d-glucose and/or mannose (1.6 mol), 2,3,6-tri-O-methyl-d-mannose (5.6 mol), and 2,3,6-tri O-methyl-d-glucose (21.2 mol) were found in the glucomannan. Periodate and Smith-degradation studies substantiate the results of methylation analysis on the xylan. A glucose: mannose ratio of 3:1 for the glucomannan, however, suggests that this fraction may be an aggregate of true glucomannan and glucan or degraded cellulose.     

Temperature dependence-independence of antifreeze turnover in Eurosta solidaginis (Fitch)

Temperature effects on antifreeze metabolism were investigated in two populations (northern and southern) of the golden rod gallfly, Eurosta solidaginis. Sorbitol production was temperature dependent and was triggered by short-term exposure to < +10°C. The maximal rate sorbitol synthesis occurred at 0°C. For both populations, sorbitol was rapidly catabolized during warm acclimation at +20°C. During the first 12 h of warm acclimation, sorbitol levels decreased by 36% (19.7 ± 0.6) to 12.6 ± 1.2 μg/mg) and by 83% (to 3.3 ± 1.7 μg/mg) after 48 h in the northern population. The southern population decreased sorbitol levels 64% (11.8 ± 0.69 to 4.2 ± 0.62) after 48 h. The southern population resynthesized more sorbitol than did the northern population upon re-exposure to 0°C. Glycerol levels increased linearly during the experimental period independent of temperature.     

Structural features of two amyloids from the hemi-cellulosic fraction of field-bean (Dolichos lablab) hulls

Two amyloid-type fractions were isolated from field-bean (Dolichos lablab) hulls by 10% alkali extraction followed by acetylation and solvent fractionation. The major, chloroform-insoluble fraction and a minor, chloroform-soluble fraction were found to be homogeneous in sedimentation analysis and molecular-sieve chromatography. The polysaccharides contained xylose and glucose in various proportions. Methylation analysis, periodate oxidation, Smith degradation, oxidation by chromium trioxide, and oligosaccharide studies indicated a new type of structure for the major fraction (glucose:xylose ratio of 1.9:1) in that it had a backbone of (1→4)-linked β-d-glucose residues interspersed with single or multiple residues of (1→4)-linked β-d-xylose, and to which some single d-xylosyl groups are attached through O-6 of d-glucose. In contrast, the minor fraction (glucose:xylose ratio of 1:3.7) had a backbone of (1→4)-linked β-d-xylose interspersed with (1→4)-β-d-glucose and having a side chain of d-xylose, attached through O-6 of d-glucose. The third fraction was found to be a mixture of linear (1→4)-d-glucan and (1→4)-d-xylan.     

The production of T-cell growth factor (TCGF) in vivo in sheep

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000–20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Fatty acid composition of the lipids of Puffinus tenuirostris (Temminck) in relation to its diet

The fatty acid composition of lipids isolated from the depot fat, stomach contents, and proventricular oil of adult and chick Puffinus tenuirostris (Temminck) has been analysed. The diet of both adults and chicks is almost exclusively derived from the euphausiid Nyctiphanes australis Sars, and an attempt was made to determine whether dietary lipid affects the composition of depot fat, and whether individual fatty acids in the stomachs and proventricular oil can be used as markers for the origin of the diet. An apparent selectivity in the deposition of fatty acids in the fat depots can be explained by the conversion of fatty alcohols, derived from the euphausiid wax ester, into fatty acids of equivalent chain length and unsaturation. Hexadecadienoic acid appeared to be the only possible marker fatty acid from the euphausiid, but wide variations in its level limits its usefulness as a reliable index of the diet of Puffinus tenuirostris.     

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.     

A theory of charge separation,ion, electron and proton transport in photosynthetic membranes based on asymmetry of surface charges

We present a model for the light-induced charge separation, proton and ion transport across photosynthetic membranes based on an assumption of the transmembrane surface charge asymmetry. In dark equilibrium, this asymmetry gives rise to an internal membrane electric field whose direction is perpendicular to the membrane surfaces. The role of the field in the light-induced charge separation is similar to the function of the built-in electric field across a solid-state p-n junction. Light-generated free charge carriers in the membrane flow according to its direction and upon recombination on the surface give rise to an electrochemical potential difference for electrons across the membrane. The associated coupled electron-proton transport, and ion diffusion can be viewed as a response of the system to the light-induced redox and electric potential changes.     

Threshold of plasma testosterone required for normal mating activity in male sheep

The finer control of mating activity by testosterone in male sheep was investigated using the castrated ram (wether) as an experimental model. Adult wethers that had been castrated before puberty were injected with graded doses of testosterone propionate (TP) and mating behavior was assessed in standardized libido trials at various times during treatment. Doses of 1 to 2 mg TP/day elicited mounting behavior in wethers but did not result in intromission or ejaculation. On the other hand, TP doses of 4 mg/day or greater stimulated the complete mating response which included intromission and the ejaculatory reflex. The threshold dose of TP required for complete mating activity (4 mg/day) produced plasma testosterone levels which were lower than those normally observed in intact rams. The results of this study indicate that the behavioral aspects (arousal mechanisms) of mating in rams have a lower testosterone threshold than intromission and ejaculation (consummatory mechanisms). Also, since complete mating activity was stimulated in wethers having relatively low plasma testosterone levels, this may explain why there is no apparent relationship between plasma testosterone and mating drive in intact rams.     

Purification and physicochemical properties of an extra-cellular cycloamylose (cyclodextrin) glucanotransferase from Bacillus macerans

An extracellular cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) from Bacillus macerans was purified to homogeneity by adsorption on starch, ammonium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of 67,000 and consisted of one polypeptide chain. The isoelectric point was pH 5.4. Temperature and pH optima were 60° and 5.45.8, respectively. The purified enzyme was quite stable at 50° (pH 6.0), but lost ≈80% of its activity at 60° for 30 min (pH 6.0). Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 5.7 mg/ml.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.