Investigation into the use of Aspergillus oryzae S1 nuclease in the presence of solvents which destabilize or prevent DNA secondary structure: Formaldehyde,formamide, and glyoxal

The activity of Aspergillus oryzae S₁ nuclease in solutions which destabilize DNA secondary structure was investigated. S₁ nuclease is able to degrade single-stranded DNA in the presence of various concentrations of formaldehyde, formamide, and glyoxal. It is further shown that S₁ nuclease can be used: (1) in the presence of formaldehyde to generate cleavage products from partially denatured duplex DNA; (2) to obtain thermal-melting profiles in the presence of formamide.     

Glycerol accumulation and water content in larvae of Limenitis archippus: Their importance to winter survival

Half-grown (third instar) larvae of the viceroy butterfly enter facultative winter diapause in response to short-day photoperiod after constructing tubular silk hibernacula in the basal portions of partly eaten willow leaves. Larval water content soon decreases from 80% to about 55%. No detectable quantities of glycerol occur in diapausing larvae maintained at room temperature. Subjection to cold and freezing temperatures causes high levels of glycerol to accumulate (up to 1.9 M or 7.8 g%) within the larvae. These metabolic changes probably lower the supercooling points of the larval fluids and retard both nucleative and inoculative freezing. Diapause termination is not photoperiod dependent, but involves an increase in water content and glycerol breakdown. An unidentified enzyme possibly removes the phosphate group from α-glycerophosphate, thus forming glycerol in the diapausing larvae.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

Ovulatory patterns in the squirrel monkey (Saimiri sciureus)

The results of induced ovulation regimens on 95 squirrel monkeys (Saimiri sciureus) over a 7-year period are described. A distinct seasonal pattern of response was observed in the temperate climate.The pretreatment of squirrel monkeys with progesterone slightly suppresses the total number of follicles showing growth in response to the FSH but results in a significantly higher number of animals showing at least one ovulation.No significant differences were noted for ovarian response based on the length of time the animal had been in captivity.The effectiveness of the ovulation induction regimen over 20 or more consecutive courses of treatment was not significantly reduced, suggesting that antibodies were not formed against the gonadotropins.     

Inhibition of active K+ transport in the tobacco hornworm (Manduca sexta) midgut after ingestion of Bacillus thuringiensis endotoxin

The effect of Bacillus thuringiensis endotoxin after ingestion on the K⁺ transport of Manduca sexta midgut is described. Direct measurements of short circuit current and transepithelial potential demonstrate that the transport is inhibited at the end of 1 hr with many of the insects exhibiting both negative short circuit current and transepithelial potential by the end of a 4-hr ingestion period.     

A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

A simple radioactive assay system is elaborated for the measurement of dihyrofolate synthetase activity in Escherichia coli. It is also applicable to Neisseria gonorrhoeae and N. meningitidis extracts. Eight oxidized and reduced pteroate analogs have been examined for inhibitory activity. The most active inhibitor was dihydrohomopteroic acid followed by dihydro-10-thiopteroic acid, dihydrofolic acid, and dihydroisopteroic acid. The enzyme appears to be incapable of binding with substrate and any of the inhibitors in their oxidized forms.     

Effects of prostaglandins E2, I2 and F2α, arachidonic acid and indomethacin on pressor responses to norepinephrine in conscious rats

The effects of prostaglandins E₂ (PGE₂), I₂ (PGI₂) and F₂α (PGF₂α), arachidonic acid and indomethacin on pressor responses to norepinephrine were examined in conscious rats. Intravenously infused PGE₂ (0.3, 1.25 μg/kg/min), PGI₂ (50, 100 ng/kg/min), PGF₂α (1.8, 5.4 μg/kg/min) and arachidonic acid (0.7, 1.4 mg/kg/min) did not change the basal blood pressure. Both PGE₂ and PGI₂ significantly attenuated pressor responses to norepinephrine, whereas PGF₂α significantly potentiated them. Arachidonic acid, a precursor of the prostaglandins (PGs), significantly attenuated pressor responses to norepinephrine. Since the attenuating effect of arachidonic acid was completely abolished by the pretreatment with indomethacin (5 mg/kg), arachidonic acid is thought to exert an effect through its conversion to PGs. On the contrary, intravenously injected indomethacin (0.2–5.0 mg/kg) facilitated pressor responses to norepinephrine in a dose-related manner without any direct effect on the basal blood pressure. These results suggest that endogenous PGs may participate in the regulation of blood pressure by modulating pressor responses to norepinephrine in conscious rats.     

Production and perception of pheromones by the beetle Tribolium confusum

Adults of Tribolium confusum secrete two pheromones. The first, produced by the male, is attractive to both sexes and the second, produced by the female, is attractive to the male only. Pheromone production and perception was studied in relation to habituation, beetle age, time of day and previous mating. A living source of each pheromone habituates the responding beetles, the male pheromone habituating more strongly; female pheromone habituates only in the absence of the male pheromone. Habituation to one pheromone was always accompanied by an enhanced response to the other.Five days after emergence, production of male pheromone reaches a peak that is maintained. Production of female pheromone peaks after 3 days. Both sexes are responsive to male pheromone immediately upon eclosion, males reaching maximum response at 14 days, females at 8 days. Males are also responsive to female pheromone upon eclosion reaching maximum response at 8 days; female response to female pheromone is imperceptible. Males but not females display a 24 hr rhythm in pheromone production. Mated beetles did not differ significantly from unmated beetles in their ability to perceive pheromones. Alteration in male pheromone production after mating was detected by females but not males; this pheromone may, therefore, act as both a sex and aggregation pheromone.     

pH dependence of the oxidation-reduction potential of cytochrome c2

The pH dependence of the spectra and of the oxidation-reduction potential of three cytochromes c₂, from Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodomicrobium vannielii, were studied. A single alkaline pK was observed for the spectral changes in all three ferricytochromes. In Rps. capsulata cytochrome c₂ this spectroscopic pK corresponds to the pK observed in the dependence of oxidation-reduction potential on pH. For the other two cytochromes the oxidation-reduction potential showed a complex dependency on pH which can be fitted to theoretical curves involving three ionizations. The third ionization corresponds to the ionization observed in the spectroscopic studies but the first two occur without changes in the visible spectra.The possible structural bases for these ionizations are discussed.     

S-Adenosylmethionine,cyclopropane fatty acid synthase,and the production of lactobacillic acid in Lactobacillus plantarum

S-Adenosylmethionine (AdoMet) levels in Lactobacillus plantarum were found to increase concomitantly with the production of membrane cyclopropane fatty acids under normal growth conditions. This increase in AdoMet did not occur when the pH of the culture medium (initially pH 6.5) was not allowed to fall (pH 4 or lower) during growth. When the culture medium was maintained at pH 6.5, cyclopropane fatty acid synthesis also remained low. While the activity of cyclopropane fatty acid synthase is increased as the pH decreases, the activity of AdoMet synthetase is largely unaffected by the variation of pH of the culture medium. The production of cyclopropane fatty acids is also dependent upon continued protein synthesis; in the presence of chloramphenicol cyclopropane fatty acid synthase activity is decreased, resulting in a lowered production of cyclopropane fatty acids. A dramatic increase in AdoMet levels occurs in the presence of chloramphenicol. It is proposed that AdoMet levels, in conjunction with cyclopropane fatty acid synthase activities, regulate cyclopropane fatty acid synthesis in L. plantarum.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.     

Comparison of the transposon-like structures encoding clindamycin resistance in Bacteroides R-plasmids

The R-plasmids pBF4, pBFTM10, and pBI136 encode transmissible clindamycin resistance (Ccr) in Bacteroides spp. These plasmids are distinct replicons but the regions implicated in Ccr share some homology and appear to have a transposon-like structure. To better understand the mechanism of dissemination and to locate the Ccr determinant(s), the genetic and structural properties of the Ccr regions of each plasmid were compared and contrasted. For this work a single EcoRI restriction fragment containing the Ccr region from each plasmid was cloned into pBR322 in Escherichia coli. Results of restriction mapping and heteroduplex experiments showed that the pBF4 EcoRI-D and pBFTM10 EcoRI-B fragments shared more than 90% base sequence homology but that the EcoRI-C fragment of pBI136 had diverged significantly. The pBI136 fragment also did not confer tetracycline resistance in E. coli as shown for the pBF4 EcoRI-D fragment (D.G. Guiney, P. Hasegawa, and C. E. Davis, 1984, Plasmid 11, 248-252). Heteroduplex experiments showed that the pBI136 EcoRI-C and pBF4 EcoRI-D fragments shared a 1.2-kb region of homology attributed to a directly repeated sequence which bounds the Ccr region. Southern hybridization studies indicated that an additional 0.85 kb of the pBI136 EcoRI-C fragment was homologous to the EcoRI-D fragment of pBF4. This region was characterized by its sequential restriction endonuclease sites for HindIII, AvaII, and DdeI, and it is proposed that the Ccr gene(s) resides in this area.     

Ornithine decarboxylase in Phycomyces: In vitro and in vivo properties

The properties of ornithine decarboxylase from Phycomyces blakesleeanus were examined. Enzyme from mycelial cultures was extracted and purified approximately 70-fold. The apparent molecular weight is 96K. The Michaelis constants with respect to ornithine and pyridoxal 5′-phosphate are 90 and 0.37 μm, respectively. Putrescine is a potent competitive inhibitor with a K_i of 75 μm. Exposure of ornithine decarboxylase to sulfhydryl-modifying reagents resulted in a rapid inhibition of activity. In vivo addition of putrescine produced characteristic decreases in cellular ornithine decarboxylase activity. Light stimulation of dark-adapted mycelial cultures also decreased cellular ornithine decarboxylase activity.     

An effect of mate fertility on the attractiveness and oviposition rates of mated Drosophila melanogaster females

Mate fertility has a strong influence on the sexual receptivity of mated Drosophila melanogaster females, but an effect of mate fertility on the attractiveness of mated females has not previously been demonstrated. We compared the declines in attractiveness over the first 10 h after mating for females mated to fertile (XY) males and those mated to sterile (XO) males, and found a significant effect of mate fertility. A large and significant decrease in attractiveness, that is the same for both XY- and XO-mated females, is evident during the first 4 h after mating. However, a further decline in attractiveness occurs between 4 and 6 h after mating for XY-mated females, but not until between 6 and 10 h after mating for females with sterile mates. Thus, XY-mated females are significantly less attractive than XO-mated females at 6–8 h post-mating, but not at any other time. A sharp increase in oviposition rates for both types of mated females is associated with the decline in attractiveness that occurs between 4 and 10 h after mating.     

Purification of bovine liver microsomal NADH-cytochrome b5 reductase using affinity chromatography

Microsomal NADH-cytochrome $\text{b}{}_5$ reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × $\text{g}$ microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome $\text{b}{}_5$ is also obtained.     

Subcellular localization and some properties of the adenylate cyclase activity of the yeast, Saccharomyces cerevisiae

Saccharomyces cerevisiae was grown in the presence of 5% (w.v) Glucose and converter to protoplasts. The total particulate material obtained from lysed protoplasts was fractionated by sucrose density gradient ultracentrifugation and the distribution of adenylate cyclase throughout the gradient determined. Adenylate cyclase activity was found to be larger associated whith intracellular particulate fractions. Little activity was found in the plasma membrane-rich fraction.The adenylate cyclase activity was found to be inhibited by F^?, pyrophosphate and aminophylline, whereas glucagon, 5-hydroxytryptamine and concanavalin A were without effect.The enzymic activity appeared to be modulated by “catabolite repressors” (glucose, fructose and α-methylglucoside) as well as by acetate. A possible role for adenylate cyclase in regulating the levels of cyclic AMP in the cell during glucose repression is suggested.