Tunicamycin-resistant mutants of Bacillus amyloliquefaciens are deficient in amylase, protease and penicillinase synthesis and have altered sensitivity to antibiotics and autolysis

Mutants of Bacillus amyloliquefaciens resistant to at least 10 micrograms/ml of tunicamycin were isolated and shown to be pleiotropic. The mutants were more resistant to streptomycin, chloramphenicol, kanamycin and neomycin than was the parent strain but less resistant to penicillin G and tetracycline. They were more autolytic, presumably due to an altered cell wall. The mutants produced reduced levels of amylase, penicillinase and both metal and serine protease besides having an enhanced sporulation frequency and being more motile.     

Induction of 7-ethoxycoumarin o-deethylase activity in cultured human epithelial cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): evidence for TCDD receptor

The responsiveness of 5 human squamous cell carcinoma (SCC) lines derived from tumors of the epidermis and tongue to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was assessed by measuring the induction of the cytochrome P1-450-mediated monooxygenase activity, 7-ethoxycoumarin O-deethylase (ECOD). In 4 of the SCC lines the EC50 for this response was approximately 10(-9)M, whereas in one line the EC50 was 10(-10)M. In each of the less sensitive lines a concentration of 10(-10)M TCDD elicited less than 5% of the maximal enzyme activity. Specific binding of radiolabeled TCDD was detected in the cytosol fraction from all the SCC lines. The relative amount of receptor measured in each line correlated with maximally-induced ECOD activity. The data indicate that human cell lines derived from a target tissue for TCDD toxicity contain the TCDD receptor and show differential sensitivity to TCDD analogous to the murine strain differences in sensitivity regulated by the Ah locus.     

Effect of temperature on the protein and nucleic acid content of thermotolerant yeasts

Summary The effect of growth at 30°, 35° and 40° on the biomass yield and on the nucleic acid and protein content of twelve isolates of yeast has been studied. Although yields of 41.6% and a true protein content of 34% were obtained, each of the strains exhibited a lower yield and protein content at 40° than at the lower temperatures. Nucleic acid levels were also reduced at 40°.     

Penicillin amidohydrolase productivity of locally isolated bacterial species

Penicillin amidohydrolase productivity of four locally isolated bacterial species is described. Organisms were identified asEscherichia coli, Pseudomonas aeruginosa, Sarcina lutea andBacillus megaterium. Highest enzyme productivity of 3.2 U/mL with a corresponding dry cell mass of 4.5 g/L was recorded fromS. lutea.     

Uptake and binding of cadmium and mercury to metallothionein in rat hepatocyte primary cultures

The administration of inorganic Cd and Hg in vivo has been shown to result in markedly different metal concentrations in rat liver. Primary cultures of rat hepatocytes were utilized to gain insight into the dispositional differences between these chemically similar metals. Hepatocyte monolayer cultures were exposed to several concentrations of Cd or Hg (3, 10 and 30μm) in serum-containing medium for 30min. The cells were then washed and incubated in fresh medium for the remainder of the experiment. Hepatocytes exposed to Cd accumulated significantly more metal than hepatocytes exposed to equimolar concentrations of Hg. In cells exposed to 3μm-Cd there was an initial loss of Cd from the hepatocytes when placed in fresh medium, followed by a gradual re-uptake of metal, concomitant with increased binding to metallothionein. In hepatocytes exposed to 3 and 10μm-Cd, 87 and 77% of the intracellular Cd was bound to metallothionein within 24h. Loss of Hg from hepatocytes pulsed with 30μm-Hg was also observed upon the addition of fresh medium and continued for the duration of the experiment. No time-dependent increase in Hg binding to metallothionein was observed. A maximum of about 10% of the intracellular Hg was found associated with metallothionein in hepatocytes exposed to 30μm-Hg. Studies utilizing [³⁵S]cysteine incorporation indicated significant increases in the amount of metallothionein synthesized in hepatocytes exposed to 3 and 10μm-Cd (300% of control value) and 30μm-Hg (150% of control value) 24h after metal pulsing. Time-course studies revealed a 6–12h lag in metallothionein synthesis, followed by a significant elevation in [³⁵S]cysteine incorporation into metallothionein between 12 and 24h. These studies suggest that (a) isolated hepatocytes differentiate between Cd and Hg and preferentially accumulate the former, and (b) Cd strongly stimulates the induction of, and preferentially binds to, metallothionein, whereas Hg induces weakly, and does not preferentially bind to, metallothionein.     

The effect of inhibition of prostaglandin synthesis on ovarian hypertrophy in the rat.

Aspirin and indomethacin, inhibitors of prostaglandin biosynthesis, were utilized to determine the role of prostaglandins (PGs) in ovarian weight gain in rats following unilateral ovariectomy or treatment with PMSG. After unilateral ovariectomy, the compensatory ovarian hypertrophy was 185-0% compared with 139-8% and 97-5% in rats treated with indomethacin and aspirin, respectively. The adrenal weights in rats treated with aspirin were also reduced significantly. Administration of PGE2 or PGF2alpha with aspirin reversed the effect of aspirin on the adrenals but had no effect on the ovarian weight. Indomethacin and aspirin treatment of animals injected with PMSG also reduced the ovarian weight gain. If 100 mug PGE2 were given twice daily, this effect was reversed in both groups but thrice daily administration had no effect on rats receiving aspirin. In PMSG-treated rats, 100 mug PGF2alpha twice daily did not reverse the effect of indomethacin and aspirin, and actually enhanced the effect of aspirin.     

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at −20°C, in as little as 10–20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.     

Role of protein kinase C in NADPH oxidase derived O2-mediated regulation of KV-LVOCC axis under U46619 induced increase in [Ca]i in pulmonary smooth muscle cells

Treatment of bovine pulmonary smooth muscle cells with the TxA₂ mimetic, U46619 stimulated [Ca²⁺]_i, which was inhibited upon pretreatment with apocynin (NADPH oxidase inhibitor). Pretreatment with cromakalim (K_V channel opener) or nifedepine (L-VOCC inhibitor) inhibited U46619 induced increase in [Ca²⁺]_i, indicating a role of K_V-LVOCC axis in this scenario. Neither cromakalim nor nifedepine inhibited U46619 induced increase in NADPH oxidase activity, suggesting that the NADPH oxidase activation is proximal to the K_V-LVOCC axis in the cells. Pretreatment with calphostin C (PKC inhibitor) markedly reduced U46619 induced increase in NADPH oxidase activity and [Ca²⁺]_i in the cells. Calphostin C pretreatment also markedly reduced p^47phox phosphorylation and translocation to the membrane and association with p^22phox, a component of Cyt.b₅₅₈ of NADPH oxidase in the membrane. Overall, PKC plays an important role in NADPH oxidase derived O₂⁻-mediated regulation of K_V-LVOCC axis leading to an increase in [Ca²⁺]_i by U46619 in the cells.