Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.     

两种菌株来源的glyA基因的克隆、表达及酶活性检测

采用PCR方法,分别从大肠杆菌和嗜热链球菌基因组DNA中扩增获得glyA基因,分别克隆入载体pET-28 a(+)中并进行表达,分离和纯化得到两种不同来源的SHMT,分别检测两种SHMT的逆向酶活。比较来源于大肠杆菌K12与嗜热链球菌AS1.2471中的glyA基因表达的丝氨酸羟甲基转移酶(SHMT)的活性,以获得高活性的SHMT。结果成功获得两种菌中的glyA基因,并表达出具有较高活性的SHMT,其中嗜热链球菌中glyA基因表达出的SHMT的酶活性大约为大肠杆菌的两倍。从嗜热链球菌中克隆表达的SHMT具有更高的催化活性及良好的工业应用前景。     

曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice

The CTG repeat involved in myotonic dystrophy is one of the most unstable trinucleotide repeats. However, the molecular mechanisms underlying this particular form of genetic instability—biased towards expansions—have not yet been completely elucidated. We previously showed, with highly unstable CTG repeat arrays in DM1 transgenic mice, that Msh2 is required for the formation of intergenerational and somatic expansions. To identify the partners of Msh2 in the formation of intergenerational CTG repeat expansions, we investigated the involvement of Msh3 and Msh6, partners of Msh2 in mismatch repair. Transgenic mice with CTG expansions were crossed with Msh3- or Msh6-deficient mice and CTG repeats were analysed after maternal and paternal transmissions. We demonstrated that Msh3 but not Msh6 plays also a key role in the formation of expansions over successive generation. Furthermore, the absence of one Msh3 allele was sufficient to decrease the formation of expansions, indicating that Msh3 is rate-limiting in this process. In the absence of Msh6, the frequency of expansions decreased only in maternal transmissions. However, the significantly lower levels of Msh2 and Msh3 proteins in Msh6 -/- ovaries suggest that the absence of Msh6 may have an indirect effect.     

Purification and some properties of β-N-Acetyl-D-glucosaminidase from viscera of green crab (<Emphasis Type="Italic">Scylla serrata</Emphasis>)

β-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with K_m of 0.424 ± 0.012 mM and V_max of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na⁺ and K⁺ have no effects on the enzyme activity; Mg²⁺ and Ca²⁺ slightly activate the enzyme, while Ba²⁺, Zn²⁺, Mn²⁺, Hg²⁺, Pb²⁺, Cu²⁺, and Al³⁺ inhibit the enzyme to different extents.     

COX-2 contributes to the maintenance of flow-induced dilation in arterioles of eNOS-knockout mice

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 microM), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by approximately 51% and approximately 58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by approximately 57%. The residual portion of the response was abolished by additional administration of Nomega-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice.     

Importance of assemblage‐level thinning: A field experiment in an alpine meadow on the Tibet plateau

Question: Which fraction of the decrease in species richness under fertilization can be explained by assemblage‐level thinning? Location: An alpine meadow on the eastern Tibet plateau. Methods: 60‐m² plots were randomly assigned to a control or one of four levels of ammonium phosphate fertilizer. Treatments were repeated for three years. The effect of as semblage‐level thinning was decided based on similarity in quadrats within and between fertilizing levels, bootstrap simulation based on random thinning of the high density (low production, low fertility) quadrats and correlation of species’ biomass in low fertility and high fertility. Results: Fertilization increased production, reduced species richness and reduced density of individuals. Heavily fertilized quadrats are more similar in species composition in 2000 but less similar in 2001 and 2002. Rarefaction showed that a decrease in density can account for 32.3‐42.9% decrease of species richness, but the simulated species richness is always significantly higher than the observed one. When production and species richness are similar at two levels of fertilization, species biomass in the higher fertility treatment is positively correlated with biomass at lower fertility. When the two fertilizer levels differed in production and species richness, there was no evidence of correlation in species biomass, suggesting that assemblage level thinning cannot explain all the loss of species. Conclusion: Although a decrease in density could explain much of the decrease (up to 42.9%) in species richness when this alpine meadow was fertilized, other important mechanisms such as interspecific competition cannot be ignored. Future studies should investigate the effect of assemblage level thinning on species diversity, and search for mechanisms responsible for a decrease in diversity.     

IBA浓度、扦插时间对江西杜鹃和百合花杜鹃扦插生根的影响

为探明有鳞大花亚组杜鹃扦插生根的最佳IBA浓度和扦插时间,该研究以江西杜鹃、百合花杜鹃为材料,分别采用腐叶土+河沙(1:1)、泥炭+珍珠岩+蛭石(3:1:1)基质,开展了4个IBA浓度和4个扦插时间的生根试验.结果表明:IBA浓度对除老叶留存数外的所有指标有显著影响,其中100 mg·L-1 IBA处理生根率、新梢长最大,腐烂率最低,其它指标也表现良好,为最佳生根浓度;50 mg·L-1 IBA处理根幅、新梢率最大,但不定根数最少,效果其次;200 mg·L-1 IBA处理促进根系生长,但生根率较低、特别是显著抑制新梢发育;对照处理生根效果最差.扦插时间对所有生根指标均有显著影响,早春(04-18)木质化硬枝扦插除老叶留存数较差外,其它指标均表现极佳,为最适扦插时间;秋季(10-19)半木质-木质化过渡枝扦插效果其次;夏季(06-21)嫩枝及(08-16)半木质化枝生根效果极差,不宜进行扦插育苗.物种、基质对生根指标也有显著影响,百合花杜鹃扦插生根能力强于江西杜鹃,泥炭+珍珠岩+蛭石(3:1:1)基质生根效果优于腐叶土+河沙(1:1).该研究结果首次发现早春新梢萌发前采用木质化硬枝扦插可以显著提高两种杜鹃的生根效果,为该亚组杜鹃的扦插育苗提供了科学依据.     

Primulina maciejewskii sp. nov. (Gesneriaceae) from Guangdong,China

Primulina maciejewskii F. Wen, R.L. Zhang & A.Q. Dong, a new species of Primulina (Gesneriaceae) from Guangdong, China, is described and illustrated. It is similar to P. lobulata and P. danxiaensis in leaf and flower shape, but can be distinguished by bract shape, peduncle, pedicel, and bract size, filament and anther indumentum, and number of bracts, staminodes and stigma.