Early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate

To improve efficiency and assess variation in nuclear transfer techniques in non-human primates, we investigated the following factors: type of donor cell, interval between enucleation and cell injection, activation after electrical pulsing and cytokinesis inhibitors. An average of 16.4 oocytes were recovered from 91 retrievals; however, 15 (14%) additional retrieval attempts yielded no oocytes due to a failure of follicular stimulation. Oocyte maturation rates at 36, 38 and 40 h post-hCG were 46.2, 52.6 and 61.2%, respectively. The MII spindle could be seen clearly using polarized microscopy in 89.1% (614/689) of oocytes. Nuclei were seen in 42% of the NT couplets, 53% of those cleaved to the 2-cell stage and 63% of the 2-cell embryos developed to the 8-cell stage by Day 3. There was no difference in the occurrence of nuclear formation between couplets created using fibroblasts or cumulus cells, although embryos were more reliably produced with fibroblasts. The interval (2, 3 and 4 h) between enucleation and cell injection did not affect NT efficiency. Ethanol treatment after electrical pulses yielded more 2-cell NT embryos than did treatment with ionomycin, but the frequency of nuclear formation and development to the 8-cell stage was not different. Treatment of couplets with cycloheximide and cytochalasin B for 5 h after activation had no impact on NT efficiency.     

Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis

A mitochondrial glycerol-3-phosphate (G-3-P) shuttle that channels cytosolic reducing equivalent to mitochondria for respiration through oxidoreduction of G-3-P has been extensively studied in yeast and animal systems. Here, we report evidence for the operation of such a shuttle in Arabidopsis thaliana. We studied Arabidopsis mutants defective in a cytosolic G-3-P dehydrogenase, GPDHc1, which, based on models described for other systems, functions as the cytosolic component of a G-3-P shuttle. We found that the gpdhc1 T-DNA insertional mutants exhibited increased NADH/NAD+ ratios compared with wild-type plants under standard growth conditions, as well as impaired adjustment of NADH/NAD+ ratios under stress simulated by abscisic acid treatment. The altered redox state of the NAD(H) pool was correlated with shifts in the profiles of metabolites concerning intracellular redox exchange. The impairment in maintaining cellular redox homeostasis was manifest by a higher steady state level of reactive oxygen species under standard growth conditions and by a significantly augmented hydrogen peroxide production under stress. Loss of GPDHc1 affected mitochondrial respiration, particularly through a diminished capacity of the alternative oxidase respiration pathway. We propose a model that outlines potential involvements of a mitochondrial G-3-P shuttle in plant cells for redox homeostasis.     

Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.     

Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from <Emphasis Type="Italic">Escherichia coli</Emphasis>

A comparative evaluation of five different cell-disruption methods for the release of recombinant hepatitis B core antigen (HBcAg) from Escherichia coli was investigated. The cell disruption techniques evaluated in this study were high-pressure homogenization, batch-mode bead milling, continuous-recycling bead milling, ultrasonication, and enzymatic lysis. Continuous-recycling bead milling was found to be the most effective method in terms of operating cost and time. However, the highest degree of cell disruption and amounts of HBcAg were obtained from the high-pressure homogenization process. The direct purification of HBcAg from the unclarified cell disruptate derived from high-pressure homogenization and bead milling techniques, using batch anion-exchange adsorption methods, showed that the conditions of cell disruption have a substantial effect on subsequent protein recovery steps.     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

氧合血红蛋白Fe-O2键合态 ab initio 研究

本文报道用自旋非限制Hartree-Fock方法(spin unrestricted Hartree-Fock method简写UHF)对氧合血红蛋白的Fe-O₂键合态进行ab initio研究。结果表明Fe^(Ⅱ)、Oc和Or的Mulliken布居值分别为24.18、8.19和7.64。这说明氧合血红蛋白的Fe-O₂键合态不发生电子转移。研究模型所得到的频率与实验频率基本一致。研究结果不支持Weiss提出的Fe³⁺O₂^-模型,为解释血红蛋白传输氧的作用机理提供了新的理论依据。     

Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences

Previous studies have shown that gene therapy with inducible nitric oxide synthase (iNOS) protects against myocardial infarction at 3 days after gene transfer. However, the long-term effects of iNOS gene therapy on myocardial ischemic injury and cardiac function are unknown. To address this issue, we used a recombinant adenovirus 5 (Ad5) vector (Av3) with deletions of the E1, E2a, and E3 regions, which enables long-lasting recombinant gene expression for at least 2 mo due to lack of inflammation. Mice received intramyocardial injections in the left ventricular (LV) anterior wall of Av3/LacZ (LacZ group) or Av3/iNOS (iNOS group); 1 or 2 mo later, they were subjected to myocardial infarction (30-min coronary occlusion followed by 4 h of reperfusion). Cardiac iNOS gene expression was confirmed by immunoblotting and activity assays at 1 and 2 mo after gene transfer. In the iNOS group, infarct size (percentage of risk region) was significantly reduced (P < 0.05) both at 1 mo (24.2 +/- 3.4%, n = 6, vs. 48.0 +/- 3.6%, n = 8, in the LacZ group) and at 2 mo (23.4 +/- 3.1%, n = 8, vs. 36.6 +/- 2.4%, n = 7). The infarct-sparing effects of iNOS gene therapy were as powerful as those observed 24 h after ischemic preconditioning (23.1 +/- 3.4%, n = 10). iNOS gene transfer had no effect on LV function or dimensions up to 8 wk later (echocardiography). These data demonstrate that iNOS gene therapy mediated by the Av3 vector affords long-term (2 mo) cardioprotection without inflammation or adverse functional consequences, a finding that provides a rationale for further preclinical testing of this therapy.     

Perceived Empathy of Service Providers Mediates the Association between Perceived Discrimination and Behavioral Intention to Take Up HIV Antibody Testing Again among Men Who Have Sex with Men

HIV antibody testing is a key measure of HIV prevention for men who have sex with men (MSM). The World Health Organization recommends sexually active and at-risk MSM to take up HIV antibody testing regularly. This study aimed to investigate the prevalence of behavioral intention to take up HIV antibody testing in the next six months among Hong Kong MSM who were ever-testers. An anonymous cross-sectional survey recruited 326 MSM who had taken up HIV antibody testing from gay-friendly venues and internet in Hong Kong. Of the participants, 40.8% had had unprotected anal intercourse with regular or non-regular male sex partners in the last six months; they were at risk of HIV transmission despite experience in HIV antibody testing. Only 37.2% showed a strong intention to take up HIV antibody testing again in the next six months. Adjusted analysis showed that both perceived discrimination toward Hong Kong MSM (AOR = .60, 95% CI: .36–.98) and the CARE Measure assessing perceived empathy of service providers (AOR = 1.05, 95% CI: 1.02–1.08) were significantly associated with intention for retesting. Perceived discrimination, however, became statistically non-significant (AOR = .68, 95% CI: .41–1.14), when both CARE Measure and perceived discrimination entered into the adjusted model. It is warranted to increase HIV retesting rate by removing perceived discrimination and reducing the negative effect of perceived discrimination through enhancement of empathy of service providers.     

Revealing Pathway Dynamics in Heart Diseases by Analyzing Multiple Differential Networks

Development of heart diseases is driven by dynamic changes in both the activity and connectivity of gene pathways. Understanding these dynamic events is critical for understanding pathogenic mechanisms and development of effective treatment. Currently, there is a lack of computational methods that enable analysis of multiple gene networks, each of which exhibits differential activity compared to the network of the baseline/healthy condition. We describe the iMDM algorithm to identify both unique and shared gene modules across multiple differential co-expression networks, termed M-DMs (multiple differential modules). We applied iMDM to a time-course RNA-Seq dataset generated using a murine heart failure model generated on two genotypes. We showed that iMDM achieves higher accuracy in inferring gene modules compared to using single or multiple co-expression networks. We found that condition-specific M-DMs exhibit differential activities, mediate different biological processes, and are enriched for genes with known cardiovascular phenotypes. By analyzing M-DMs that are present in multiple conditions, we revealed dynamic changes in pathway activity and connectivity across heart failure conditions. We further showed that module dynamics were correlated with the dynamics of disease phenotypes during the development of heart failure. Thus, pathway dynamics is a powerful measure for understanding pathogenesis. iMDM provides a principled way to dissect the dynamics of gene pathways and its relationship to the dynamics of disease phenotype. With the exponential growth of omics data, our method can aid in generating systems-level insights into disease progression.