从酵母表达时间序列估计基因调控网络

基因调控网络是生命功能在基因表达层面上的展现。用组合线性调控模型、调控元件识别和基因聚类等方法 ,从基因组表达谱解读酵母在细胞周期与环境胁迫中的基因调控网络。结果表明 ,细胞在不同环境条件下会调整基因调控网络。在适应的环境下 ,起主要作用的是细胞生长和增殖有关的基因调控网络 ;而在响应环境胁迫时 ,细胞会再规划调控网络 ,抑制细胞生长和增殖相关的基因 ,诱导跟适应性糖类代谢与结构修复相关的基因 ,还可能启动减数分裂产生孢子。分别从细胞周期和环境胁迫响应相关基因中 ,搜索到转录因子Mcm1结合位点TT CC T GGAAA ,和Dal82在尿囊素代谢途径相关基因上的结合位点TGAAAAWTTT。从而 ,从酵母表达时间序列估计基因调控网络是可行的 ,与至今已知的实验观察相当吻合     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

Genetic Structure and Aggressiveness of Rhizoctonia solani AG1‐IA,the Cause of Sheath Blight of Rice in Southern China

One hundred and eighty isolates of Rhizoctonia solani AG1‐IA, the causal agent of rice sheath blight, were obtained from six locations in southern China. The genetic structure of R. solani isolates was investigated using random amplified polymorphic DNA (RAPD) markers, and a considerable genetic variation among R. solani isolates was observed. Most of the genetic diversity was distributed within populations, rather than among them. The distribution pattern of the genetic variation of R. solani appears to be the result of high gene flow (Nm) and low‐genetic differentiation among populations. The aggressiveness of R. solani was visually assessed by rice seedlings of five different cultivars in the glasshouse. All isolates tested were found to induce significantly different levels of disease severity, reflecting considerable variation in aggressiveness. The isolates were divided into highly virulent, moderately virulent and weakly virulent groups, and the moderately virulent isolates were dominant in R. solani population. No significant correlation was observed among the genetic similarity, pathogenic aggressiveness and geographical origins of the isolates. Information obtained from this study may be useful for breeding for improved resistance to sheath blight.     

Relationship between chemokine (C–X–C motif) ligand 12 gene variant (rs1746048) and coronary heart disease: Case–control study and meta-analysis

The goal of our study is to evaluate the contribution of CXCL12 rs1746048 (hg19, chr10:44775574) to the risk of CHD in Han Chinese, and to summarize its role in CHD through meta-analysis of existing studies among various ethnic groups. Significant association is observed between rs1746048-C and an increased risk of CHD in Han Chinese (χ² = 5.41, df = 1, P = 0.02). Post hoc analysis reveals an even stronger association of rs1746048 with the risk of CHD for subjects aged 65 years or older (genotype: χ² = 8.39, df = 2, P = 0.015; allele: χ2 = 9.13, df = 1, P = 0.003, odd ratio (OR) = 1.91, 95% confidential interval (CI) = 1.25–2.91). A break down analysis by gender shows that rs1746048 is likely a CHD risk factor under the recessive model in males (CC + CT versus TT: P = 0.05, χ² = 3.59, df = 1, OR = 1.72, 95% CI = 1.00–3.04). In addition, a meta-analysis of ten studies among over 107,000 individuals confirms that rs1746048 is a risk factor of CHD (P < 0.0001, OR = 1.12, 95% CI = 1.09–1.15) and this agrees with the findings of our case–control study in Han Chinese.     

Differentiation of human adipose‐derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca²⁺ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.     

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae

S-Adenosyl-l -methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l -methionine is generally used, the low-cost dl -methionine is more suitable as the substrate for industrial production of SAM. However, d -methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl -methionine, intracellular conversion of d -methionine to l -methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d -amino acid-N-acetyltransferase, d -methionine was accumulated in vivo and no N-acetyl-d -methionine production was observed. Further, codon-optimized d -amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l -phenylalanine dehydrogenase gene (l -PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d -methionine to l -methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d -methionine. Using the recently developed CRISPR tools, the DAAO and l -PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl -methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl -methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl -methionine.     

Behaviour of photosynthetic products associated with growth and grain production in the rice plant

Summary A study of the behaviour of the photosynthetic products assimilated at different growth stages was conducted in the field and in the greenhouse using C¹⁴ tracer.In general, the assimilated carbon is translocated to and accumulates in the growing organs. The carbon assimilated at the maximum tiller number stage is distributed mostly to the lower leaves. The carbon assimilated at the booting stage is distributed mostly to the spikelet, certain leaf sheaths and culms. The carbon accumulated in the form of carbohydrates in the leaf sheaths and the culm before flowering is retranslocated to the panicle after flowering. However, because of the consumption by respiration, the efficiency of this type of carbohydrate in grain production is not very high. The carbon assimilated after flowering accumulated mostly and efficiently in the brown rice.The release of the assimilated carbon as CO₂ is most intense immediately after assimilation. Thirty-five to 60 per cent of the assimilated carbon is consumed through respiration under the conditions of this experiment. As the carbon, which is in the form of sugars, rapidly changes to other forms, and also is consumed by respiration, the consumption declines rapidly. The retention percentage of assimilated carbon decreases as mutual shading increases.The large proportion of carbon released through respiration indicates the importance of studies on the significance of respiration in relation to growth.A portion of the thesis for the Master of Science degree submitted by Mr. Shen Lian to the Graduate School, University of the Philippines, College of Agriculture.     

Lipid-Dependent Modulation of Ca2+ Availability in Isolated Mossy Fiber Nerve Endings

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca²⁺, since exocytosis requires Ca²⁺ and a potentiation of Ca²⁺ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca²⁺ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca²⁺ accumulation was reduced by inhibitors of phospholipase A₂ and voltage-sensitive Ca²⁺ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A₂ was also indicated by the ability of staurosporine to reduce the Ca²⁺ accumulation induced by arachidonic acid or the phospholipase A₂ activator melittin. Similarly, the synergistic facilitation of evoked Ca²⁺ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca²⁺ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.     

Association of PON1, P2Y12 and COX1 with Recurrent Ischemic Events in Patients with Extracranial or Intracranial Stenting

Background and Purpose

Short-term combined use of clopidogrel and aspirin improves cerebrovascular outcomes in patients with symptomatic extracranial or intracranial stenosis. Antiplatelet non-responsiveness is related to recurrent ischemic events, but the culprit genetic variants responsible for the non-responsiveness have not been well studied. We aimed to identify the genetic variants associated with poor clinical outcomes.

Methods

Patients with symptomatic extracranial or intracranial stenosis scheduled for stenting and receiving dual antiplatelets (clopidogrel 75 mg and aspirin 100 mg daily) for at least 5 days before intervention were enrolled. Ischemic events including recurrent transient ischemic attack, stroke, myocardial infarction, and vascular-related mortality within 12 months follow-up were recorded. We examined the influence of genetic polymorphisms on treatment outcome in our patients.

Results

A total of 268 patients were enrolled into our study and ischemic events were observed in 39 patients. For rs662 of paraoxonase 1 (PON1), allele C was associated with an increased risk of ischemic events (OR = 1.64, 95%CI = 1.03–2.62, P = 0.029). The A-allele carriers of rs2046934 of P2Y12 had a significant association with adverse events (OR = 2.01, 95%CI = 1.10–3.67, P = 0.041). The variant T-allele of cyclooxygenase-1 (COX1) rs1330344 significantly increased the risk of recurrent clinical events (OR = 1.85, 95%CI = 1.12–3.03, P = 0.017). The other single nucleotide polymorphism (SNP) had no association with ischemic events.

Conclusions

PON1, P2Y12 and COX1 polymorphisms were associated with poorer vascular outcomes. Testing for these polymorphisms may be valuable in the identification of patients at risk for recurrent ischemic events.