PRIMARY STRUCTURE OF ALLATOSTATIN 4, A NEVROPEPTIDE INHIBITOR OF JUVENILE HORMONE SYNTHESIS

Abstract Isolation and identification of allatostatin 4 (AST 4) from crude brain extracts of female Diploptera punctata are described. Synthetic analogues of allatostatin 4 truncated from the N-terminal were evaluated to gain some knowledge of structure-activity correlation. AST 4 reversibly inhibits the biosynthesis of JH in vitro . AST 4 has the following sequence: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂. This neurohormone (AST 4) has no sequence similarity with any known neuropeptide from other organisms. Synthetic AST 4 as well as truncation fragments, including two with the five and six amino acids terminal residues deleted showed in vitro activity distinguishable from that of the native allatostatin.     

Screening of rice cultivars for Cr-stress response by using the parameters of seed germination,morpho-physiological and antioxidant analysis

Rice is the most important crop for the majority of population across the world with sensitive behavior toward heavy metals such as chromium (Cr) in polluted regions. Although, there is no information on the Cr resistance phenotyping in rice. Herein, two different groups of rice cultivars (normal, and hybrid) were used, each group with 14 different rice cultivars. Firstly, seed germination analysis was conducted by evaluating various seed germination indices to identify the rice cultivars with greatest seed germination vigor. Furthermore, exposure of chromium (Cr) toxicity to 28 different rice varieties (NV1-NV14, HV1-HV14) caused noticeable plant biomass reduction. Subsequently, NV2, NV6, NV10, NV12, NV13 (normal type), HV1, HV4, HV8, and HV9 (hybrid types) were pragmatic as moderately sensitive varieties, while NV3, NV4, NV9, and NV14 (normal type), HV3, HV6, HV7, and HV13 were observed as moderately tolerant. Although, NV7, and HV10 were ranked most sensitive cultivars, and NV11, and HV14 were considered as most tolerant varieties as compared to the other rice (both groups) genotypes. Afterward, Cr induced reduction in chlorophyll pigments were significantly lesser in HV14 relative to NV11, NV7, and especially HV10, and as a result HV14 modulated the total soluble sugar level as well as reduced ROS accumulation, and MDA contents production by stimulating the antioxidant defense mechanism conspicuously which further reduced the electrolyte leakage as well. Our outcomes provide support to explore the Cr tolerance mechanism in cereal crops as well as knowledge about rice breeding with increased tolerance against Cr stress.     

A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure.     

Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation

We have previously described regulation of focal adhesion kinase (FAK) by its amino-terminal FERM-like domain through an autoinhibitory interaction with its kinase domain (Cooper, L. A., Shen, T. L., and Guan, J. L. (2003) Mol. Cell. Biol. 23, 8030-8041). Here we show that the first two subdomains of the FERM-like domain are independently capable of inhibiting phosphorylation of FAK in trans. We characterized several point mutations within the first subdomain of the FERM-like domain and find that mutation of Lys-38 to alanine results in a FAK mutant that is strongly hyperphosphorylated when expressed in mammalian cells, and promotes increased phosphorylation of the FAK substrate paxillin. A second mutation of Lys-78 to alanine results in a FAK mutant that is underphosphorylated, but can be activated by extracellular matrix stimuli. Like deletion of the amino terminus itself the K38A mutation is phosphorylated in suspension. The Delta375 truncation mutant of FAK is strongly phosphorylated both when Tyr-397 is mutated to phenylalanine, and in the presence of the Src inhibitor, PP2, suggesting that removal of the amino terminus can render FAK Src independent. This is in contrast to the K38A mutant that is not phosphorylated in the Y397F background, and which shows decreased phosphorylation in the presence of the Src inhibitor PP2, suggesting that regulation of FAK by Src is a secondary step in its activation. The K38A mutation weakens the interaction between the amino terminus of FAK and its own kinase domain, and disrupts the ability of the amino terminus to inhibit the phosphorylation of FAK in trans. The K38A mutation of FAK also increases the ability of FAK to promote cell cycle progression and cell migration, suggesting that hyperphosphorylation of this mutant can positively affect FAK function in cells. Together, these data strongly suggest a role for the first FAK subdomain of the FERM domain in its normal regulation and function in the cell.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

胰高血糖素样肽-2对小鼠小肠缺血/再灌注损伤的保护作用

目的:观察胰高血糖素样肽-2(GLP-2)对缺血/再灌注损伤小鼠小肠的保护效应.方法:采用肠缺血/再灌注(I/R)模型,将32只小鼠随机分为4组(n=8)假手术(Sham)组、I/R组、I/R GLP-2保护组和I/R 谷氨酰胺(GLN)阳性对照组.光镜观察小肠黏膜形态学改变.检测小肠绒毛高度和隐窝深度;小肠组织二胺氧化酶(DAO)活性;肠系膜淋巴结(MLN)细菌易位率.结果:与假手术组相比,I/R组部分小肠绒毛坏死脱落,绒毛高度下降,隐窝变浅(P＜0 01);小肠组织DAO活性降低(P＜0.01);MLN细菌易位率增加(P＜0.05).与I/R组比,GLP-2组肠绒毛损害明显减轻,DAO活性回升(P＜0.01),细菌易位率回降(P＜0.05).结论:GLP-2对缺血/再灌注损伤小鼠小肠的形态结构及肠屏障功能具有保护作用.     

Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway

Diarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.     

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.