Regulation of endothelial nitric oxide synthase by protein kinase C

Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M., Titani, K., and Taniguchi, H. (1996) Biochemistry 35, 14651-14658]. Here we report that bovine eNOS protein is phosphorylated at Thr497 in the calmodulin-binding domain by PKC both in vitro and in vivo, and that the phosphorylation negatively regulates eNOS activity. A specific antibody that recognizes only the phosphorylated form of the enzyme was raised against a synthetic phosphopeptide corresponding to the phosphorylated domain. The antibody recognized eNOS immunoprecipitated with anti-eNOS antibody from the soluble fraction of bovine aortic endothelial cells, and the immunoreactivity increased markedly when the cells were treated with phorbol 12-myristate 13-acetate. PKC phosphorylated eNOS specifically at Thr497 with a concomitant decrease in the NOS activity. Furthermore, the phosphorylated eNOS showed reduced affinity to calmodulin. Therefore, PKC regulates eNOS activity by changing the binding of calmodulin, an eNOS activator, to the enzyme.     

Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, gamma-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1(tr/tr)), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1(tr/tr) animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1(tr/tr) mice are sensitive to ionizing radiation (gamma-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.     

Crystal structure of human DJ-1, a protein associated with early onset Parkinson's disease

We report the crystal structure at 1.8-A resolution of human DJ-1, which has been linked to early onset Parkinson's disease. The monomer of DJ-1 contains the alpha/beta-fold that is conserved among members of the DJ-1/ThiJ/PfpI superfamily. However, the structure also contains an extra helix at the C terminus, which mediates a novel mode of dimerization for the DJ-1 proteins. A putative active site has been identified near the dimer interface, and the residues Cys-106, His-126, and Glu-18 may play important roles in the catalysis by this protein. Studies with the disease-causing L166P mutant suggest that the mutation has disrupted the C-terminal region and the dimerization of the protein. The DJ-1 proteins may function only as dimers. The Lys to Arg mutation at residue 130, the site of sumoylation of DJ-1, has minimal impact on the structure of the protein.     

植物性雌激素genistein对豚鼠乳头肌的电生理效应

应用细胞内微电极技术,观察了genistein(GST)对豚鼠乳头肌的电生理效应。结果显示：（1）GST（10-100μmol/L)浓度依赖地缩短正常乳头肌动作电位时程;（2）对部分去极化乳头肌,GST（50μmol/L)除缩短动作电位时程外,还使动作电位幅值和超射值降低,零相最大上升速度减慢;（3）预先应用一氧化氮合酶抑制剂L-NNA（5mmol/L)。不影响GST（50μmol/L)的电生理效应;（4）单独应用17β-雌二醇（E2,5μmol/L)或GST（10μmol/L)时,动作电位各参数无明显变化。而预先应用同剂量的GST再加入E2,则动作电位时程缩短,结果提示,GST可能通过非NO途径抑制Ca^2 内流,从而影响豚鼠乳头肌电生理效应,并与E2有加强或协同效应。     

Ontogenetic Behavior and Migration of Chinese Sturgeon, Acipenser sinensis

The Chinese sturgeon, Acipenser sinensis, is an anadromous protected species that presently only spawns in the Yangtze River. Using laboratory experiments, we examined the behavioral preference of young Chinese sturgeon to physical habitat (water depth, illumination intensity, substrate color, and cover) and monitored their downstream migration. Hatchling free embryos were photopositive, preferred open habitat, and immediately upon hatching, swam far above the bottom using swim-up and drift. Downstream migration peaked on days 0–1, decreased about 50% or more during days 2–7, and ceased by day 8. Days 0–1 migrants were active both day and night, but days 2–7 migrants were most active during the day. After ceasing migration, days 8–11 embryos were photonegative, preferred dark substrate and sought cover. Free embryos developed into larvae and began feeding on day 12, when another shift in behavior occurred–larvae returned to photopositive behavior and preferred white substrate. The selective factor favoring migration of free embryos upon hatching and swimming far above the bottom may be avoidance of benthic predatory fishes. Free embryos, which must rely on yolk energy for activity and growth, only used 19 cumulative temperature degree-days for peak migration compared to 234 degree-days for growth to first feeding larvae, a 1:12 ratio of cumulative temperature units. This ratio suggests that sturgeon species with large migratory embryos, like Chinese sturgeon, which require a high level of energy to swim during migration, may migrate only a short time to conserve most yolk energy for growth.     

Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.     

Inheritance of S(f)-RNase in Japanese apricot ( Prunus mume) and its relation to self-compatibility

Self-compatible cultivars of Japanese apricot ( Prunus mume Shieb. et Zucc.), a tree species that normally shows S-RNase-based self-incompatiblity, have a horticultural advantage over self-incompatible cultivars. Inheritance of self-compatibility and a common S(f)-RNase allele that is observed in self-compatible cultivars was investigated using progenies from controlled crosses. Total DNAs were isolated from the parents and progenies of seven crosses that included at least one self-compatible cultivar as a parent. These DNAs were PCR-amplified with the Pru-C2 and PCE-R primer pair to determine S-haplotypes of the parents and progenies. A novel S-haplotype, S(8), was found. In all crosses examined, the S(f)-RNase gene was inherited from either the seed or pollen parent as a pistil S-allele in a non-functional S-haplotype. Self-compatibility of about 20 trees each from reciprocal crosses of 'Benisashi ( S(7) S(f))' and 'Shinpeidayu ( S(3) S(f))', and 26 selections from 16 different crosses was tested by pollination and pollen-tube growth studies. Cosegregation of the S(f)-RNase allele and self-compatibility was confirmed with all but selection 1K0-26 ( S(3) S(7)). Selection 1K0-26 ( S(3) S(7)) that originated from 'Benisashi ( S(7) S(f))' x 'Koshinoume ( S(3) S(f))' appeared to be self-compatible even without the S(f)-RNase allele. The possible role of pollen- S, a presumably existing pollen component of gametophytic self-incompatibility, is discussed.