αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-α_vβ₃ antibodies prevent cell adhesion to FGF-2. Also, purified human α_vβ₃ binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-α_vβ₃ monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with α_vβ₃ integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1–4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1–5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1–3F) and FGFR1-Y463/583/585/766F mutant (FGFR1–4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1–3F, and FGFR1–4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)₂, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK₂ activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK_1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.     

Repercussions of the COVID-19 pandemic on athletes: a cross-sectional study

The COVID-19 pandemic has presented significant challenges and implications for the sports community. Thus, this study aimed to describe the prevalence of COVID-19 in Brazilian athletes and identify the epidemiological, clinical, athletic, life and health factors associated with the disease in these individuals. A cross-sectional study was performed involving 414 athletes from 22 different sports using an online questionnaire from August to November 2020. The association between the athletes’ characteristics and COVID-19 was evaluated using a logistic regression model. The prevalence of COVID-19 was 8.5%, although only 40% of athletes reported having been tested. Being under 27 years of age (3-fold), having children (~5-fold), having a teammate test positive for COVID-19 (2.5-fold), and smoking (14-fold) were associated with a possible higher risk of disease. Almost 20% of athletes self-reported musculoskeletal injuries during the period of the pandemic that was studied. Athletes with a university education (P = 0.02), a profession other than sports (P < 0.001), those from a low-income family (P = 0.01), and public health system users (P = 0.04) were significantly less frequently tested for COVID-19, whereas international competitors, athletes who received a wage, and athletes who had a teammate who tested positive for COVID-19 were 2-, 3-, and 15-fold more likely to be tested for COVID-19, respectively. Approximately 26% of the athletes who tested negative or were untested reported more than three characteristic COVID-19 symptoms, and 11% of athletes who tested positive for COVID-19 were asymptomatic. The identification of modifiable (have children, smoking, and teammates positively tested) and non-modifiable (age under 27 years) factors related to COVID-19 in athletes can contribute to implementing surveillance programmes to decrease the incidence of COVID-19 in athletes and its negative impacts in sports.     

马鹿茸血酶解肽体内免疫功能及抗氧化功能关系的研究

本文分别选用由木瓜蛋白酶水解天山马鹿茸血获得的肽Ⅰ、肽Ⅱ以及原血为受试样品,研究天山马鹿茸血及其酶解肽对小鼠免疫功能和抗氧化功能的影响,其中肽Ⅰ、肽ⅡDH分别为18．1％、12．2％时,清除OH&#183;能力最强,清除率为50．8％。分别测定了脏器指数,脾细胞增殖实验,血清中溶血素和抗体生成细胞的含量以及小鼠血清总抗氧化能力、SOD活力和MDA含量。结果表明：与对照组相比较,肽Ⅱ组能极显著提高小鼠体液和细胞免疫功能,加强小鼠的抗氧化功能（P〈0．01或P〈0．05）。此外,具有较强抗氧化活性的肽显示出了很强的免疫活性（P〈0．05）。     

Blood Biochemistry of Sea Turtles Captured in Gillnets in the Lower Cape Fear River,North Carolina,USA

ABSTRACT Mortality due to fisheries interactions has been implicated as a contributor to population decline for several species of sea turtle. The incidental capture of sea turtles in the coastal gillnet fisheries of North Carolina, USA, has received much attention in recent years, and mitigation measures to reduce sea turtle mortality due to gillnet entanglement are a high priority for managers and conservationists. Efforts to evaluate effects of gillnet entanglement on sea turtle populations are complicated by the lack of information on health status of turtles released alive from nets and postrelease mortality. We obtained blood samples from green (Chelonia mydas) and Kemp's ridley (Lepidochelys kempii) sea turtles captured in gillnets for 20–240 minutes to assess the impacts of gillnet entanglement on blood biochemistry and physiological status. We measured concentrations of lactate, corticosterone, ions (Na⁺, K⁺, Cl^-, P, Ca²⁺), enzymes (lactate dehydrogenase [LDH], creatine phosphokinase [CPK], aspartate aminotransferase [AST]), protein, and glucose in the blood and also performed physical examinations of turtles to document external indicators of health status (injuries, lethargy, muted reflexes). We evaluated the effects of entanglement time on blood biochemistry and to look for correlations between blood biochemistry and results of the physical examinations. We observed a significant increase in blood lactate, LDH, CPK, phosphorus, and glucose with increased entanglement time. Alterations in blood biochemistry were generally associated with a decline in health status as indicated by results of the physical examination. Although entanglement time plays an important role in determining the health status of sea turtles upon release from a gillnet, our results suggest that factors such as the depth and severity of entanglement may also have an effect on health status of turtles and the probability of postrelease survival. We were unable to set a maximum unattended gillnet soak time to minimize impacts on captured sea turtles, and therefore recommend that fisheries managers continue to enforce the net attendance regulations currently in place in the lower Cape Fear River, North Carolina, during the summer months.     

Zebrafish embryo, a tool to study tumor angiogenesis

Zebrafish (Danio rerio) represents a powerful model system in cancer research. Recent observations have shown the possibility to exploit zebrafish to investigate tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Experimental models have been established in zebrafish adults, juveniles, and embryos, each one with its own advantages and disadvantages. Novel genetic tools and high resolution in vivo imaging techniques are also becoming available in zebrafish. It is anticipated that zebrafish will represent an important tool for chemical discovery and gene targeting in tumor angiogenesis. This review focuses on the recently developed tumor angiogenesis models in zebrafish, with particular emphasis to tumor engrafting in zebrafish embryos.     

急性心肌梗死患者冠脉搭桥术前中性粒细胞-淋巴细胞比率与围术期心肌损伤相关分析

目的：探讨急性心肌梗死患者冠脉搭桥（CABG）术前中性粒细胞．淋巴细胞比率（NLR）与围术期心肌损伤的关系,为,临床CABG围术期心肌保护提供参考依据。方法：选取2012年1月至2012年6月于首都医科大学附属北京安贞医院因急性心肌梗死接受冠脉搭桥手术（CABG）患者210例,收集术前血常规及术后肌钙蛋白I（cTnI）？Life酸激酶同工酶（CK-MB）,计算NLR;采用四分位法根据NLR水平将患者分为四组,比较各组cTnI及CK—MB峰值,多元逐步回归分析NLR与cTnI及CK-MB峰值的相关性。结果：随着NLR水平升高,高血压病史和射血分数〈50％患者比例逐渐增多;白细胞计数、术后CK-MB及cTnI峰值、术后血肌酐值均逐渐增加;多元逐步回归分析显示,NLR、WBC分别与cTnI峰值呈正相关（r=0．526,r=0．186,P〈0．05）。结论：术前NLR、WBC与cTnI峰值呈正相关,NLR可能是反应急性心肌梗死患者冠脉搭桥围术期心肌损伤的良好标志物。     

Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat.

Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.     

Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.     

Early liver alterations induced by the sex-dependent hepatocarcinogen beta-blocker ZAMI 1305

Liver alterations occurring after 1, 6 or 10 days of treatment with the hepatocarcinogen beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butyl-amino-propan-2-ol (ZAMI 1305) were studied in male and female Wistar rats. In agreement with its sex-dependent oncogenicity, ZAMI 1305 administration causes DNA damage in the liver of the female but not of the male rat, with the only exception of 2 out of 4 males treated for 6 days. In female rat, the amount of DNA damage increases from 1 to 6 days of treatment, being unchanged at 10 days; a small portion of DNA is however damaged. ZAMI 1305 administration to female rat induces also: (i) an increase of the relative liver weight, of the DNA and RNA synthesizing activity; (ii) a decrease of the number of hepatocytes in mitosis; (iii) a minimal oval cell hyperplasia. When the same parameters were studied in ZAMI 1305-treated male rats, they were unaffected or changed to a less extent in respect to female rats.