Role of Cathepsin G in the Degradation of Glyceraldehyde-3-Phosphate Dehydrogenase Triggered by 4-Hydroxy-2-Nonenal in U937 Cells

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.

Emerging role of autophagy in colorectal cancer:Progress and prospects for clinical intervention

Autophagy is a physiological mechanism in which cells degrade themselves and quickly recover the degraded cell components.Recent studies have shown that autophagy plays an important role in the occurrence,development,treatment,and prognosis of colorectal cancer.In the early stages of colorectal cancer,autophagy can inhibit the production and development of tumors through multiple mechanisms such as maintaining DNA stability,inducing tumor death,and enhancing immune surveillance.However,as colorectal cancer progresses,autophagy may mediate tumor resistance,enhance tumor metabolism,and other pathways to promote tumor development.Therefore,intervening in autophagy at the appropriate time has broad clinical application prospects.This article summarizes the recent research progress of autophagy and colorectal cancer and is expected to provide new theoretical basis and reference for clinical treatment of colorectal cancer.

The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes

Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms （DXS1, DXS2）. In tomato （Solanum lycopersicum）, the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.

Co-ordinated combination of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway in Escherichia coli to promote L-tryptophan production

In this study,phosphoenolpyruvate and erythrose-4-phosphate are efficiently supplied by collaborative design of Embden-Meyerhof-Parnas(EMP)pathway and pentose phosphate(PP)pathway in Escherichia coli,thus increasing the L-tryptophan production.Firstly,the effects of disrupting EMP pathway on L-tryptophan production were studied,and the results indicated that the strain with deletion of phosphofructokinase A(i.e.,E.coli JW-5ΔpfkA)produced 23.4±2.1 g/L of L-tryptophan production.However,deletion of phosphofructokinase A and glucosephosphate isomerase is not conducive to glucose consumption and cell growth,especially deletion of glucosephosphate isomerase.Next,the carbon flux in PP pathway was enhanced by introduction of the desensitized glucose-6-phosphate dehydrogenase(zwf)and 6-phosphogluconate dehydrogenase(gnd)and thus increasing the L-tryptophan production(i.e.,26.5±3.2 g/L vs.21.7±1.3 g/L)without obviously changing the cell growth(i.e.,0.41 h^(-1) vs.0.44 h^(-1))as compared with the original strain JW-5.Finally,the effects of co-modifying EMP pathway and PP pathway on L-tryptophan production were investigated.It was found that the strain with deletion of phosphofructokinase A as well as introduction of the desensitized zwf and gnd(i.e.,E.coli JW-5 zwf243 gnd361ΔpfkA)produced 31.9±2.7 g/L of L-tryptophan,which was 47.0%higher than that of strain JW-5.In addition,the glucose consumption rate of strain JW-5 zwf243 gnd361ΔpfkA was obviously increased despite of the bad cell growth as compared with strain JW-5.The results of this study have important reference value for the following application of metabolic engineering to improve aromatic amino acids producing strains.

No Significant Association between PIK3CA Mutation and Survival of Esophageal Squamous Cell Carcinoma:A Meta-analysis

The prognostic value of phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha(PIK3CA) in patients with esophageal squamous cell carcinoma(ESCC) is controversial. We aimed to investigate the prognostic significance of PIK3CA mutation in patients with ESCC. EMBASE, Pub Med, and Web of Science databases were systematically searched from inception through Oct. 3, 2016. The hazard ratios(HRs) and 95% confidence intervals(CI) were calculated using a random effects model for overall survival(OS) and disease-free survival(DFS). Seven studies enrolling 1505 patients were eligible for inclusion of the current meta-analysis. Results revealed that PIK3CA mutation was not significantly associated with OS(HR: 0.90, 95% CI: 0.63–1.30, P=0.591), with a significant heterogeneity(I2=65.7%, P=0.012). Additionally, subgroup analyses were further conducted according to various variables, such as types of specimen, the sample size, technique and statistical methodology. All results suggested that no significant relationship was found between PIK3CA mutation and OS in patients with ESCC. For DFS, there was no significant association between PIK3CA mutation and DFS in patients with ESCC(HR: 1.00, 95% CI=0.47–2.11, P=0.993, I2=73.7%). Publication bias was not present and the results of sensitivity analysis were very stable in the current meta-analysis. Our findings suggest that PIK3CA mutation has no significant effects on OS and DFS in ESCC patients. More well-designed prospective studies with better methodology for PIK3CA assessment are required to clarify the prognostic significance of PIK3CA mutation in ESCC patients.

Monolayers Langmuir-Blodgett Films of Synthetic Artificial Mimic Molecules That Resemble the Following Tetraether Lipids on Silicon Wafers

This study investigated the behavior and molecular organization of synthetic artificial mimic molecules that resemble the following tetraether lipids: di-O-hexadecyl-glycero-3-phosphatidyl-glycerol (DHGPG) and bis-4-dodecylphenyl-12-phosphate. These molecules were analyzed using Langmuir film balance, ellipsometry and atomic force microscopy. The monolayer Langmuir-Blodgett films of DHGPG and bis-4-dodecylphenyl-12-phosphate were stable on the solid surface silicon wafers. The ellipsometry and AFM results showed that monolayers Langmuir-Blodgett films of DHGPG and bis-4-dodecylphenyl-12-phosphate were present, and the thickness of the observed films varied from 1.2 - 5.0 nm.

Phosphoinositide and phospholipid phosphorylation and hydrolysis pathways<br/>—Organophosphate and organochlorine pesticides effects<br>

Phospholipid and phosphoinositide phosphorylation pathways have been shown to be of crucial importance on producing lipid mediators. The earlier findings reported on lipid molecules playing roles in different metabolic pathways used to assign them the exclusive role of second messenger generators. Several researchers have recently described how direct interaction of phospholipids and phosphoinositides with molecules or organelles, without the need for producing second messenger molecules, is responsible for their mechanism of action. Organophosphate and organochlorine pesticide toxicity mechanisms have been extensively studied in relation to their well known effects on cholinesterase activities and on the alterations of electric activity in the nervous system of different organisms respectively. There is little but consistent evidence that some compounds, including in both groups of pesticides, are also able to interact with phospholipid and phosphoinositide phosphorylation pathways in several organisms and tissues. The present review consists of an actualization of basic research on phospholipid and phosphoinositide phosphorylation and hydrolysis pathways, as well as a description of some reported evidences for the effects of the above mentioned pesticides on them.

Inflammatory pathways in pathological neovascularization in retina and choroid: a narrative review on the inflammatory drug target molecules in retinal and choroidal neovascularization

Objective:We review inflammatory drug targets in retinal and choroidal neovascularization(NV)in narrative manner.Background:Vascular remodeling and angiogenesis are processes typically associated with wound-healing mechanisms intended to minimize ischemia and maintain tissue homeostasis.In the eye,however,these actions primarily deteriorate tissue homeostatic recovery,and could even contribute to the progress of severe conditions,e.g.,blindness.Angiogenesis in diabetic retinopathy(DR)and age-related macular degeneration(AMD)is the primary cause of vision loss in working-age and elderly populations.Current treatment of anti-vascular endothelial growth factor(VEGF)agents has limited action efficacy,working in less than 50%patients.Understanding cellular and molecular networks associated in retinal vascular remodeling may provide an insight to develop novel therapeutic strategies.Methods:Here,we highlight ocular cells-endothelial,mural,retinal pigment epithelium(RPE),glial and macrophages,as well as inflammatory molecules-such as the complement system,stromal derived factor-1,chemokine CXC receptor-4,inflammasome,interleukin-18,programed cell death ligand-1,insulin-like growth factor(IGF)and sphigosin-1-phosphate receptor,associated with retinal and choroidal NV,and discuss their recent and future therapeutic approaches.Conclusions:A deeper understanding on pathogenesis,pathobiology including ocular immunobiology of retinal and choroidal NV will pave the way to expand and overleap the current therapeutic approach.

一种新的抗菌药物靶标蛋白质(3,4-二羟基-2-丁酮-4-磷酸合成酶)的克隆、表达、纯化及酶活性鉴定

【目的】核黄素(vitamin B12,riboflavin)是辅因子黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)和黄素单核苷酸(flavin mononucleotide,FMN)的前体物,对生物体的生物合成至关重要。如果细菌不能够从外界摄取足够的黄素(flavin)就需要自身合成核黄素以维持菌体的生存与增殖。3,4-二羟基-2-丁酮-4-磷酸合成酶(3,4-Dihydroxy-2-butanone-4-phosphate synthase,DHBPs)为核黄素生物合成途径中关键酶之一。在镁离子存在的情况下,DHBPs将5-磷酸核酮糖(ribulose-5-phosphate,Ru5P)转换成3,4-二羟基-2-丁酮-4-磷酸(3,4-dihydroxy-2-Bu-tanone-4-Pho-sphate,DHBP)和甲酸盐(formate),生成的DHBP为核黄素合成的必需原料之一。人类没有合成核黄素的相关途径,因此细菌参与合成核黄素的DHBPs等相关酶就有望成为抗菌药物作用的靶位点。本课题通过对肺炎链球菌的DHBPs进行克隆表达纯化与酶学性质鉴定,为开展其三维结构的解析和抗菌药物设计提供重要的工作基础。【方法】利用PCR技术扩增DHBPs基因,构建重组表达载体pW28-DHBPs。将其转入大肠杆菌(Escherichia coli)BL21(DE3)中表达,用Ni离子亲和层析及离子交换(DEAE)纯化获得有活性的DHBPs后,进行酶学性质鉴定。【结果】酶切和测序证实成功构建了质粒pW28-DHBPs,在E.coli BL21中表达了可溶性DHBPs,纯化后获得了纯度为95%的靶蛋白质,经分子筛分析DHBPs在溶液中以二聚体形式存在。对DHBPs进行酶学性质分析表明,在25℃、pH为7.5和Mg2+存在的情况下,DHBPs具有将5-磷酸核酮糖转换成DHBP和甲酸盐的活性。【结论】第一次成功克隆并在E.coli BL21中表达了一种肺炎链球菌合成核黄素的相关酶—DHBPs,纯化后的重组DHBPs具有较好的5-磷酸核酮糖分解活性,这为解析其三维结构和基于结构进行的新一代抗菌药物设计提供重要的工作基础。

Curcumin Alleviates Hyperandrogenism and Promotes Follicular Proliferation in Polycystic Ovary Syndrome Rats:Insightson IRS1/PI3K/GLUT4 and PTEN Modulations

Objective:To explore the effect of curcuminon the insulin receptor substrate1(IRS1)/phosphatidylinositol-3-kinase(PI3K)/endometrial expression of glucose 4(GLUT4)signalling pathway and its regulator,phosphatase and tensin homolog(PTEN),in a rat model of polycystic ovarian syndrome(PCOS).Methods:PcoS model was induced by letrozole intragastric administration.Sprague-Dawleyrats were randomized into 4groups according to a random number table:(1)control group;(2)PcoS group,which was subjected to PCOS and received vehicle;(3)curcumin group,which was subjected to PCoS and treated with curcumin(200 mg/kg for 2 weeks);and(4)curcumin+LY294002 group,which was subjected to PCOS,and treated with curcumin and LY294002(a specific PI3K inhibitor).Serum hormone levels(17β-estradiol,follicle stimulating hormone,luteinizinghormone,progesterone,and testosterone)were measured by enzyme linked immunosorbentassay,and insulin resistance(IR)was assessed using the homeostasismodel assessment of IR.Ovarian tissues were stained with haematoxylin and eosin for pathological and apoptosis examination.Expression levels of key transcriptional regulators and downstream targets,including IRS1,Pl3K,protein kinase B(AKT),GLUT4,and PTEN,were measured via reverse transcription polymerase chain reaction and Western blot,respectively.Results:The Pcos group showed impaired ovarian morphology and function.Compared with the PCoS group,curcumin treatment exerted ovarioprotective effects,down-regulated serum testosterone,restored IR,inhibited inflammatory cell infiltration in ovarian tissues,decreased IRS1,PI3K,and AKT expressions,and up-regulated GLUT4 and PTEN expressions in PCOS rats(P<0.05orP<0.01).In contrast,IRS1,PI3K,AKT,and PTEN expression levelswerenot significantly different between PCOS and curcumin+LY294002 groups(P>0.05).Conclusion:The beneficial effects of curcumin on PCOS rats included the alteration of serum hormone levels and recovery of morphological ovarian lesions,in which,PTEN,a new target,may play a role in regulating the IRS1/PI3K/GLUT4 pathway.

Functional Cooperativity of Enzymes of Phosphoinositide Conversion According to Synergistic Effects on Pectin Secretion in Tobacco Pollen Tubes

The Arabidopsis phosphoinositide kinases PI4Kβ1 and PIP5K5 have been implicated in the control of directional vesicle trafficking underlying polar tip growth in pollen tubes. PI4Kβ1 and PIP5K5 catalyze key consecutive steps of phosphoinositide conversion, and it appears obvious that phosphatidylinositol-4-phosphate formed by PI4Kβ1 might act as a substrate for phosphatidylinositol-4,5-bisphosphate formation by PIP5K5. However, this hypothesis has not been experimentally addressed and distinct localization patterns of PI4Kβ1, PIP5K5, and also PI-synthases （PIS） generating phosphatidylinositol suggest additional complexity. Here, the synergistic functionality of enzymes of phosphoinositide conversion was assessed. In tobacco and Arabidopsis pollen tubes, phosphoinositides influence the apical secretion of pectin, and increased pectin deposition results in characteristic morphological alterations. Catalytically active and dominant negative variants of PI4Kβ1 and PIP5K5 were systematically co-expressed in tobacco pollen tubes and the incidence of morphologies related to enhanced pectin secretion was evaluated. The data support a proposed functional interplay of PI4Kβ1 and PIP5K5 at the trans-Golgi network, mediating directional vesicle trafficking. Co-expression experiments additionally including PIS isoforms, PIS1 or PIS2, indicate that pectin secretion is synergistically mediated by PI4Kβ1 and PIPSK5 acting on Ptdlns formed by PIS2 rather than PIS1. Possible ramifications for the preferential channeling of phosphoinositide intermediates between particular isoforms of PI pathway enzymes are discussed.