Purification of cytochrome P-450 from polychlorinated biphenyl-treated crab-eating monkeys: high homology to a form of human cytochrome P-450

Cytochrome P-450, designated as P-450-MK2, was purified to an electrophoretic homogeneity from polychlorinated biphenyl (PCB)-treated female crab-eating monkeys. P-450-MK2 catalyzed nifedipine and nilvadipine oxidations, at a rate comparable to human P-450-HM1. The N-terminal amino acid sequence of P-450-MK2 was highly homologous to those of P-450-HM1 and NF 25. The antibodies to P-450-HM1 recognized P-450-MK2 and effectively inhibited the activity of testosterone 6 beta-hydroxylase in monkey liver microsomes. These results suggest that a form of cytochrome P-450 corresponding to human P-450-HM1 or P-450NF which belongs to the P450 III gene family is also present in liver microsomes of crab-eating monkeys.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Basic steroid saponins from aerial parts of Fritillaria Thunbergii

From the aerial parts of Fritillaria thunbergii, three glycosidal Solanum alkaloids (basic steroid saponins) were isolated together with minor     

Ultrastructural and biochemical identification of con A receptors in the desmosome

Correlated ultrastructural and biochemical methods were used to identify and localize Concanavalin A (Con A) receptors in the desmosomes of bovine epidermis. Specific carbohydrate residues were labeled with ferritin-Con A in thin sections of tissue embedded in a hydrophilic resin. Quantitative mapping of ferritin distribution in labeled desmosomes revealed that Con A receptors are localized in the intercellular zone and concentrated along the desmosomal midline or central dense stratum. Labeling was almost entirely absent when sections were treated with ferritin-Con A in the presence of 0.1 M α-methyl mannoside, a hapten-inhibitor of Con A. “Whole” desmosomes and desmosomal intercellular regions (desmosomal “cores”) were purified from bovine muzzle epidermis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals a limited number of major desmosomal protein constituents. Certain of these are glycoproteins and are greatly enriched in the core fraction. Almost all the desmosomal glycoproteins are intensely labeled when electrophoretic gels of whole desmosome or core fractions are exposed to fluorescent Concanavalin A.