Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

Engineering membrane protein overproduction in Escherichia coli

Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold.     

Actin organization and root hair development are disrupted by ethanol-induced overexpression of Arabidopsis actin interacting protein 1 (AIP1)

* Actin organization and dynamics are essential for cell division, growth and cytoplasmic streaming. Here we analyse the effects of the overexpression of Actin Interacting Protein 1 (AIP1) on Arabidopsis development. * Arabidopsis plants were transformed with an ethanol-inducible AIP1 construct and the characteristics of these plants were analysed after induction. * When AIP1 was increased to approx. 90% above wild-type values, root hair development and actin organization in all cell types examined were disrupted. * Our data demonstrate that AIP1 is a key regulator of actin organization and that its regulation is essential for normal plant cell morphogenesis.     

mbr-FPGS高效表达质粒增强Jurkat细胞对甲氨蝶呤的敏感性

叶酰多聚谷氨酸盐合成酶(Folylpolyglutamate synthetase,FPGS)是将化疗药物甲氨蝶呤(Methotrexate,MTX)转化成甲氨蝶呤多聚谷氨酸盐(MTXPG)的关键酶,其表达水平直接影响肿瘤细胞对MTX敏感性。与B细胞急性淋巴细胞白血病(B-ALL)相比,T细胞急性淋巴细胞白血病(T-ALL)细胞中FPGS表达水平低,因此对MTX不敏感。本实验室前期研究证实,位于BCL2基因3′-UTR区的一段长279 bp的DNA序列mbr具有显著的增强子效应。文章构建了含有mbr增强子样序列的FPGS表达质粒,用其转染Jurkat细胞后,分别以Westernblotting和MTT法检测FPGS表达水平及MTX对肿瘤细胞的抑制率。结果表明mbr能够显著提高FPGS表达质粒的表达水平,并有效增强Jurkat细胞对MTX的敏感性。这一结果为将基础研究结果应用于临床、提高MTX对T-ALL细胞的化疗疗效提供了新的思路。     

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.     

A novel histidine kinase gene, ZmHK9, mediate drought tolerance through the regulation of stomatal development in Arabidopsis

Plants have developed complex signaling networks to regulate biochemical and physiological acclimation, environmental signals were perceived and transmitted to cellular machinery to activate adaptive responses. Here, a novel drought responsive histidine kinase gene was identified and designated as ZmHK9. Under normal conditions, ZmHK9 was predominantly expressed in roots, and the roots of ZmHK9-OX transgenic lines are markedly hypersensitive to ABA and ethylene, as compare to wild type. Consistent with its expression induced by PEG and exogenous ABA treatment, promoter sequence of this gene possessed drought and ABA responsive element. Moreover, the transgenic plants were much less affected by drought stress and recovered quickly after rewatering, stomatal complex size and stomatal density in the transgenic plants are significantly smaller and lower than those of the wild-type plants. In addition, ABA induced stomatal closure and the stomatal aperture of ZmHK9-OX lines was smaller than that of wild type. Collectively, it can be concluded that ZmHK9 regulates root elongation, stomatal development and drought tolerance through ABA dependent signaling pathway in Arabidopsis.     

Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803

The biosynthetic pathway for the pteridine moiety of cyanopterine, as well as tetrahydrobiopterine, has been investigated in Synechocystis sp. PCC 6803. Open reading frames slr0426, slr1626, slr0078 and sll0330 of the organism putatively encoding GTP cyclohydrolase I, dihydroneopterine aldolase, 6-pyruvoyltetrahydropterine synthase and sepiapterine reductase, respectively, have been cloned into T7-based vectors for expression in Escherichia coli. The recombinant proteins have been purified to homogeneity and demonstrated to possess expected genuine activities except that of sll0330. Our result is the first direct evidence for the functional assignment of the open reading frames in Synechocystis sp. PCC 6803. Furthermore, the 6-pyruvoyltetrahydropterine synthase gene is demonstrated for the first time in prokaryotes. Based on the result, biosynthesis of cyanopterine is discussed.