Solid-phase synthesis and conformational properties of angiotensin converting enzyme catalytic-site peptides: the basis for a structural study on the enzyme-substrate interaction

The solution NMR conformational properties of two angiotensin converting enzyme (ACE) Zn catalytic-site 36-residue peptides, with the general sequence HEMGHX23EAIGDX3, synthesized through solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, is reported. The 1H resonance assignment of Zn-bound peptides is presented and the characteristic features of the NMR solution models of the two ACE Zn(II)-bound peptides are reported. The solid-state and solution structures of the ACE C-domain catalytic site are compared while biologically important structural similarities and differences of the N- and C-terminal catalytic sites are discussed. Additionally, the structural features of the ACE substrate, the angiotensin I (AI) decapeptide, are studied using NMR spectroscopy, in order to set the structural basis for the ACE-substrate interaction in solution.     

Soluble guanylyl cyclase activation by HMR-1766 (ataciguat) in cells exposed to oxidative stress

Many vascular diseases are characterized by increased levels of ROS that destroy the biological activity of nitric oxide and limit cGMP formation. In the present study, we investigated the cGMP-forming ability of HMR-1766 in cells exposed to oxidative stress. Pretreatment of smooth muscle cells with H(2)O(2) reduced cGMP production stimulated by sodium nitroprusside (SNP) or BAY 41-2272. However, pretreatment with H(2)O(2) significantly increased HMR-1766 responses. Similar results were obtained with SIN-1, menadione, and rotenone. In addition, HMR-1766 was more effective in stimulating heme-free sGC compared with the wild-type enzyme. Interestingly, in cells expressing heme-free sGC, H(2)O(2) inhibited instead of potentiated HMR-1766 responses, suggesting that the ROS-induced enhancement of cGMP formation was heme dependent. Moreover, using truncated forms of sGC, we observed that the NH(2)-terminus of the beta(1)-subunit is required for the action of HMR-1766. Finally, to study tolerance development to HMR-1766, cells were pretreated with this sGC activator and reexposed to HMR-1766 or SNP. Results from these experiments demonstrated lack of tolerance development to HMR-1766 as well as lack of cross-tolerance with SNP. We conclude that HMR-1766 is an improved sGC activator as it has the ability to activate oxidized/heme-free sGC and is resistant to the development of tolerance; these observations make HMR-1766 a promising agent for treating diseases associated with increased vascular tone combined with enhanced ROS production.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

NMR study of non-structural proteins–part III: <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and side-chain resonance assignment of macro domain from <Emphasis Type="Italic">Chikungunya</Emphasis> virus (CHIKV)

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130–190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/β/α sandwich topology with 4 α-helices and 6 β-strands was predicted by TALOS+.     

Expression, purification, and physicochemical characterization of the N-terminal active site of human angiotensin-I converting enzyme.

We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361 to Gly468, ACE-N) of human somatic angiotensin-I converting enzyme in Escherichia coli. This construct represents the N-catalytic domain including the two binding motifs and the 23 amino acid spacers as well as some amino acid residues before and after the motifs that might help in correct conformation. The overexpressed protein was exclusively localized to insoluble inclusion bodies. Inclusion bodies were solubilized in an 8-M urea buffer. Purification was carried out by differential centrifugation and gel filtration chromatography under denaturing conditions. About 12 mg of ACE-N peptide per liter of bacterial culture was obtained. The integrity of recombinant protein domain was confirmed by ESI/MS. Structural analysis using CD spectroscopy has shown that, in the presence of TFE, the ACE-N protein fragment has taken a conformation, which is consistent with the one found in testis ACE by X-ray crystallography. This purification procedure enables the production of an isotopically labeled protein fragment for structural studying in solution by NMR spectroscopy.     

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）。