Mitochondrial activation and the pyruvate paradox in a human cell line

Pyruvate promotes hyperpolarization of the inner mitochondrial membrane. However, in isolated mitochondria, pyruvate could participate in a futile cycle leading to mitochondrial depolarization. Here, we investigated this paradox in intact human cells by measuring parameters reflecting mitochondrial activation in response to 1 mM pyruvate and 5 mM glucose. NAD(P)H levels were elevated similarly by both substrates. Conversely, pyruvate induced a first transient phase of mitochondrial depolarization before the establishment of the expected sustained hyperpolarization. This correlated with kinetics of cytosolic ATP levels exhibiting a first phase decrease followed by an increase. Therefore, pyruvate transiently depolarizes mitochondria and reduces ATP in intact cells.     

The clinical significance of circulating miR-21, miR-142, miR-143, and miR-146a in patients with prostate cancer

BackgroundProstate cancer (PCa) is the most common type of solid tissue cancer among men in western countries. In this study, we determined the levels of circulating miR-21, miR-142, miR-143, miR-146a, and RNU 44 levels as controls for early diagnosis of PCa.MethodsThe circulating miRNA levels in peripheral blood samples from 43 localized PCa patients, 12 metastatic PCa (MET) patients, and a control group of, 42 benign prostate hyperplasia (BPH) patients with a total of 97 volunteers were determined the by PCR method.ResultsNo differences in the DCT values were found among the groups. In PCa and PCaMet groups the expression of miR21 and miR142 were higher compared to the BHP group. No other differences were observed among the other groups. miR21 expression in the PCa group was 6.29 folds upregulated whereas in the PCaMet group 10.84 folds up-regulated. When the total expression of miR142 is evaluated, it showed a positive correlation with mir21 and mir 146 (both p<0.001). Also, the expression of miR146 shows a positive correlation with both miR21 and miR143 (both p<0.001). Expression of miRNAs was found to be an independent diagnostic factor in patients with Gleason score, PSA, and free PSA levels.ConclusionsOur study showed that co-expression of miR21, miR-142, miR-143, and miR-146a and the upregulation of miR-21 resulted in increased prostate carcinoma cell growth. In the PCaMet group, miR21 is the most upregulated of all miRNAs. These markers may provide a novel diagnostic tool to help diagnose PCa with aggressive behavior.     

miR-143-3p促进C2C12成肌细胞分化

MicroRNAs (miRNAs) 是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用 real-time PCR 检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p 的模拟物和特异性抑制剂分别处理细胞,采用 real-time PCR 和 Western印迹分别检测成肌因子 MyoG和成肌标志基因 MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达 miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC 的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分化被显著抑制.检测miR-143-3p对MyHC各亚型表达的影响发现,miR-143-3p表达的变化并不直接影响MyHC各亚型的表达.以上结果说明, miR-143-3p在骨骼肌和成肌细胞中均有表达,能够促进C2C12成肌细胞分化,但并不直接调控MyHCs的表达.     

Baculovirus Host-Range

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified. Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range     

Circulating microRNA signatures serve as potential diagnostic biomarkers for Helicobacter pylori infection

Helicobacter pylor (H pylori), a Gram-negative, microaerobic human pathogen, has been found to be involved in many gastroduodenal diseases. Accurate diagnosis of H pylori infection is a vital part of the effective management of gastroduodenal diseases. Circulating microRNAs (miRNAs) have shown the potential to be used as noninvasive biomarkers for the diagnosis of infectious diseases. The aim of this study was to explore plasma miRNAs as noninvasive biomarkers for H pylori infection. We performed a plasma miRNA expression profile using Illumina high-throughput sequencing and validated the levels of differentially expressed miRNAs in the plasma of 63 H pylori-infected patients and 41 healthy volunteers by quantitative real-time polymerase chain reaction (qRT-PCR). The sequencing results showed that 37 miRNAs were upregulated in the H pylori-infected patients compared with that in the healthy volunteers, while six miRNAs were downregulated. qRT-PCR and receiver operator characteristic analysis suggested that the expression of miR-28-3p, miR-143-3p, miR-151a-3p, and miR-148a-3p were closely associated with H pylori infection. Therefore, the four plasma miRNA panels mentioned above could serve as promising noninvasive biomarkers of H pylori infection.     

Epitope mapping of novel monoclonal antibodies to human angiotensin I‐converting enzyme

Angiotensin I‐converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach–conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross‐reactivity, ACE mutagenesis, and competition with the previously mapped anti‐ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs–on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer''s disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue‐specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer''s disease.     

Immobilization of proteins on aldehyde-activated polyacrylamide supports

A method has been developed for the immobilization of proteins on derivatized polyacrylamide gels. Aminoethyl Bio-Gel P-150 was converted to its stable N-2,3-dihydroxypropyl derivative by borohydride reduction of the Schiff base formed with glyceraldehyde. Periodate oxidation of the modified gel provided a reactive aldehyde, which was subsequently coupled to protein by reductive amination with sodium cyanoborohydride. Coupling efficiencies were found to be >90% for concanavalin A and bovine serum albumin, and the gels contained as much as 5 and 20 mg of protein/ml of gel, respectively. Immobilized concanavalin A retained 89% of its binding capacity and was demonstrated to be chemically stable with variations in pH, and changes in concentrations of Triton X-100 and sodium dodecyl sulfate (at concentrations <0.1%). Bovine β-hexosaminidase and β-glucuronidase, higher molecular weight proteins, were also bound with retention of activity, but with less efficiency. This procedure provides an efficient method for the covalent immobilization of proteins.     

GPR143 controls ESCRT-dependent exosome biogenesis and promotes cancer metastasis

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Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α-N-1-(1-deoxyglucitol)-lysine and ?-N-1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ?-amino groups of lysine.