Chemical modification of cysteine residues is a misleading indicator of their status as active site residues in the vitamin K-dependent gamma-glutamyl carboxylation reaction

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.     

粘弹剂在不同眼球破裂伤手术中的应用

目的：分析粘弹剂在不同眼球破裂伤手术中的有效作用。方法：回顾并分析27例不同程度眼球破裂伤患者的治疗,其中包括复杂角膜裂口并虹膜脱出修复术、摘出前房异物、瞳孔区非磁性异物、虹膜根部断离,晶体损伤或脱位等。术中应用粘弹剂固定异物、充填前房及玻璃体腔。观察术后6个月的眼球形态、视力等指标。结果：术后观察最少6个月。27例眼球全部保留,24例有视力。3例因继发视网膜脱离复住2次手术失败而失明,后眼球萎缩,植入义眼。11例白内障现代囊外摘除或PHACO术后植入人工晶体,3例晶体脱位者摘除晶体后缝线植入人工晶体。11眼视力在0．5以上。无眼内炎及交感性眼炎发生。单纯前节手术10人中均有术后轻度眼压增高,但很快恢复正常。3例因伴有视网膜脱离患者术后眼压低,术后失明。结论：不同重度眼外伤后眼内容大量丢失。眼球形态政变。应用粘弹刑可为手术提供方便,也能为保全眼球并恢复视能提供机会。在前房异物取出时能有效固定异物,为异物的取出创造是好的基础。     

糖尿病伤口愈合的分子机制

伤口愈合不良是糖尿病的一个并发症,严重时可能导致截肢,已成为糖尿病患者住院率最高的疾病,但是其确切的分子机制尚不清楚。目前研究发现糖尿病发生时有多种分子和细胞功能受损,如生长因子、一氧化氮、活性氧分子、基质金属蛋白酶、m icroRNA、内皮祖细胞等,最终导致糖尿病伤口愈合不良。本文将就其近年研究的相关分子机制予以详述。     

雌激素神经保护作用机制:线粒体功能的调节

大量研究表明雌激素具有神经保护作用,但其机制尚不清楚。近年来研究提示,雌激素的神经保护作用与线粒体有着密切联系。线粒体是细胞内能量和活性氧自由基(ROS)的主要来源,对细胞内信号转导、细胞存活与死亡调节等具有十分重要的影响。在生理和病理条件下,雌激素可多方面调节线粒体功能,包括影响ATP与ROS的生成、稳定线粒体膜电位、维护细胞内钙稳态,以及调节线粒体基因和蛋白表达等。本文主要从线粒体角度综述了雌激素神经保护作用及其机制。     

Effects of Glucose, Insulin, and Supernatant from Pancreatic β-cells on Brain–Pancreas Relative Protein in Rat Hippocampus

Brain–pancreas relative protein (BPRP) is a novel protein that mainly expresses in brain and pancreas. In our previous study, we found that various stressors significantly decreased the expression of BPRP in pancreas in vivo, accompanied by changes in insulin and glucose levels, and that expression of BPRP in pancreas also decreased significantly in diabetic rats induced by Streptozocin (STZ). All these findings suggest that BPRP may be a glucose or insulin-sensitive protein. However, how the changes in insulin or glucose levels influence the expression of BPRP in hippocampus requires further study. Here, we investigated the effects of insulin or glucose on the expression of BPRP in primary cultured hippocampal neurons. We supplied hippocampal neurons with glucose, insulin, or supernatant from pancreatic β-cells, which secrete insulin into the supernatant. Our data showed that insulin had beneficial effect on the viability while no significant effect on the expression of BPRP in hippocampal neurons. On the contrary, 40 mM glucose or free glucose culture significantly decreased the expression of BPRP, while had no significant effect on the viability and apoptosis of hippocampal neurons. Further study showed that levels of insulin in the supernatant collected from pancreatic β-cells medium changed over days, and that supernatant increased the viability of hippocampal neurons, while it had no obvious effect on the expression of BPRP in hippocampal neurons. These results suggest that BPRP may be a glucose-sensitive protein.     

Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.     

NAD+-dependent sirtuin 1 and 6 proteins coordinate a switch from glucose to fatty acid oxidation during the acute inflammatory response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.     

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR₂) than on HIV‐1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR_1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR_1M more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR_1M with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR_1M and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR_1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR_1M and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR_1M partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.