Somatostatin down-regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes

Endozepines, a family of regulatory peptides related to diazepam-binding inhibitor (DBI), are synthesized and released by astroglial cells. Because rat astrocytes express various subtypes of somatostatin receptors (sst), we have investigated the effect of somatostatin on DBI mRNA level and endozepine secretion in rat astrocytes in secondary culture. Somatostatin reduced in a concentration-dependent manner the level of DBI mRNA in cultured astrocytes. This inhibitory effect was mimicked by the selective sst4 receptor agonist L803-087 but not by the selective sst1, sst2 and sst3 receptor agonists L779-591, L779-976 and L797-778, respectively. Somatostatin was unable to further reduce DBI mRNA level in the presence of the MEK inhibitor U0126. Somatostatin and the sst1, sst2 and sst4 receptor agonists induced a concentration-dependent inhibition of endozepine release. Somatostatin and the sst1, sst2 and sst4 receptor agonists also inhibited cAMP formation dose-dependently. In addition, somatostatin reduced forskolin-induced endozepine release. H89 mimicked the inhibitory effect of somatostatin on endozepine secretion. In contrast the PLC inhibitor U73122, the PKC activator PMA and the PKC inhibitor calphostin C had no effect on somatostatin-induced inhibition of endozepine release. The present data demonstrate that somatostatin reduces DBI mRNA level mainly through activation of sst4 receptors negatively coupled to the MAPK pathway, and inhibits endozepine release through activation of sst1, sst2 and sst4 receptors negatively coupled to the adenylyl cyclase/PKA pathway.     

Sensing and signalling in response to oxygen deprivation in plants and other organisms

AIMS AND SCOPE: All aerobic organisms require molecular di-oxygen (O2) for efficient production of ATP though oxidative phosphorylation. Cellular depletion of oxygen results in rapid molecular and physiological acclimation. The purpose of this review is to consider the processes of low oxygen sensing and response in diverse organisms, with special consideration of plant cells. CONCLUSIONS: The sensing of oxygen deprivation in bacteria, fungi, metazoa and plants involves multiple sensors and signal transduction pathways. Cellular responses result in a reprogramming of gene expression and metabolic processes that enhance transient survival and can enable long-term tolerance to sub-optimal oxygen levels. The mechanism of sensing can involve molecules that directly bind or react with oxygen (direct sensing), or recognition of altered cellular homeostasis (indirect sensing). The growing knowledge of the activation of genes in response to oxygen deprivation has provided additional information on the response and acclimation processes. Conservation of calcium fluxes and reactive oxygen species as second messengers in signal transduction pathways in metazoa and plants may reflect the elemental importance of rapid sensing of cellular restriction in oxygen by aerobic organisms.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

黑线姬鼠两亚种(中国东北地区及俄罗斯远东地区的东北亚种和朝鲜半岛的朝鲜亚种) 的线粒体DNA 序列缺少趋异性

为了检测黑线姬鼠两亚种(来自中国东北地区、俄罗斯远东地区的东北亚种和朝鲜半岛的朝鲜亚种)线
粒体DNA 的变异水平并确定朝鲜亚种的分类地位,我们测序分析了两亚种的线粒体DNA 细胞色素b 的部分序列
(1 054 bp)和控制区的部分序列(860 bp),并与基因库中黑线姬鼠相应的单倍型序列进行了比较。可以看出东
北亚种的序列显示出某些分异,可以被分为2 或3 个亚群,所以我们提出需要更多标本的DNA 分析来确定东北
亚种的分类地位。另外,来自韩国的朝鲜亚种的序列,与来自中国东北地区龙江和哈尔滨的东北亚种的两个亚
群相似(1 个亚群是细胞色素b 的两个单倍型,另1 个是控制区的两个单倍型),表明基于线粒体DNA 序列的遗
传多样性与现今基于形态特征对这些姬鼠的分类所得结果是不一致的。因此我们认为来自韩国的朝鲜亚种是一
个只在形态特异上不同于东北亚种的地方亚种,我们建议通过其他DNA 标记来进一步验证其亚种地位。我们还
认为朝鲜半岛不是最近的冰川期黑线姬鼠残遗种的保护区。     

Intracellular free zinc up-regulates IFN-γ and T-bet essential for Th1 differentiation in Con-A stimulated HUT-78 cells

Zinc deficiency impairs cellular immunity. Up-regulation of mRNA levels of IFN-γ, IL-12Rβ2, and T-bet are essential for Th₁ differentiation. We hypothesized that zinc increases Th₁ differentiation via up-regulation of IFN-γ and T-bet expression. To test this hypothesis, we used zinc-deficient and zinc-sufficient HUT-78 cells (a Th₀ cell line) under different condition of stimulation in this study. We also used TPEN, a zinc-specific chelator, to decrease the bioavailability of zinc in the cells. We measured intracellular free zinc, cytokines, and the mRNAs of T-bet, IFN-γ, and IL-12Rβ2. In this study, we show that in zinc-sufficient HUT-78 cells, mRNA levels of IFN-γ, IL-12Rβ2, and T-bet in PMA/PHA-stimulated cells were increased in comparison to zinc-deficient cells. Although intracellular free zinc was increased slightly in PMA/PHA-stimulated cells, Con-A-stimulated cells in 5 μM zinc medium showed a greater sustained increase in intracellular free zinc in comparison to cells incubated in 1 μM zinc. The cells pre-incubated with TPEN showed decreased mRNA levels of IFN-γ and T-bet mRNAs in comparison to cells without TPEN incubation. We conclude that stimulation of cells by Con-A via TCR, release intracellular free zinc which functions as a signal molecule for generation of IFN-γ and T-bet, and IL-12Rβ2 mRNAs required for Th₁ cell differentiation. These results suggest that zinc increase Th₁ cell differentiation by up-regulation of IFN-γ and T-bet, and IL-12Rbβ2 mRNAs.     

Omentin, a novel adipocytokine inhibits TNF-induced vascular inflammation in human endothelial cells

Omentin is a recently identified adipocytokine with insulin-sensitizing effect. While lack of omentin may be related to the pathogenesis of obesity-related cardiovascular diseases, its effect in vasculature is largely unknown. We examined effects of omentin on vascular endothelial inflammatory states. Western blotting was performed to analyze inflammatory signal transduction in cultured vascular endothelia cells. The cyclic guanosine monophosphate (cGMP) content was measured using enzyme immunoassay. Treatment of human umbilical vein endothelial cells with omentin (300 ng/ml, 20 min) induced phosphorylation of 5′-AMP-activated protein kinase (AMPK) (Thr 172) and endothelial nitric oxide (NO) synthase (eNOS) (Ser 1177). Consistently, omentin increased the cGMP level. Pretreatment with omentin (300 ng/ml, 30 min) significantly inhibited the phosphorylation of JNK as well as expression of cyclooxygenase (COX)-2 by TNF-α (5 ng/ml, 20 min–24 h). An inhibitor of JNK significantly inhibited the TNF-α-induced COX-2 expression. Inhibitory effect of omentin on TNF-α-induced COX-2 was reversed by a NOS inhibitor. The present results demonstrate for the first time that omentin plays an anti-inflammatory role by preventing the TNF-α-induced COX-2 expression in vascular endothelial cells. Omentin inhibits COX-2 induction via preventing the JNK activation presumably through activation of AMPK/eNOS/NO pathways.     

Nonspecific DNA binding and bending by HUαβ: interfaces of the three binding modes characterized by salt-dependent thermodynamics

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU_αβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (> 34 bp) gaps between bound proteins, reversibly bending it by 140^o and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around—but not wrapped on—the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Sk_i) even though their binding site sizes differ greatly; the most probable values of Sk_i on 34-bp DNA or larger DNA are − 7.5 ± 0.5. From the similarity of Sk_i values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk_i values are proposed.