Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.     

Uncoupling protein-3 (UCP3) mRNA expression in reconstituted human muscle after myoblast transplantation in RAG2-/-/gamma c/C5(-) immunodeficient mice

Uncoupling protein-3 (UCP3), which is expressed abundantly in skeletal muscle, is one of the carrier proteins dissipating the transmitochondrial electrochemical gradient as heat and has therefore been implicated in the regulation of energy metabolism. Myoblasts or differentiated muscle cells in vitro expressed little if any UCP3, compared with the levels detected in biopsies of skeletal muscle. In the present report, we sought to investigate UCP3 mRNA expression in human muscle generated by myoblast transplantation in the skeletal muscle of an immunodeficient mouse model. Time course experiments demonstrated that 7-8 weeks following transplantation fully differentiated human muscle fibers were formed. The presence of differentiated human muscle fibers was assessed by quantitative PCR measurement of the human alpha-actin mRNA together with immunohistochemical staining using specific antibodies for spectrin and the slow adult myosin heavy chain. Interestingly, we found that the expression of UCP3 mRNA was dependant on human muscle differentiation and that the UCP3 mRNA level was comparable with that found in human muscle biopsies. Moreover, the human UCP3 (hUCP3) promoter seems to be fully functional, since triiodothyronine treatment of the mice not only stimulated the mouse UCP3 (mUCP3) mRNA expression but also strongly stimulated the hUCP3 mRNA expression in human fibers formed after myoblast transplantation. To our knowledge, this is the first time that primary myoblasts could be induced to express the UCP3 gene at a level comparable of that found in human muscle fibers.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

鼎湖山南亚热带常绿阔叶林碳素积累和分配特征

研究了鼎湖山南亚热带常绿阔叶林 40 0多年林龄的锥栗 (Castanopsis chinensis)、黄果厚壳桂 (Crypto-carya concinna)和 50多年林龄的黄果厚壳桂、鼎湖钓樟 (Lindera chunii)两个群落碳素积累和分配特征。结果表明 ,两林分间植物碳素含量在不同器官和不同层中的分配格局均十分相似 ,总平均分别为 41 .980 %(锥栗、黄果厚壳桂群落 )和 40 .377% (黄果厚壳桂、鼎湖钓樟群落 )。锥栗、黄果厚壳桂群落生态系统碳总贮量为 2 4 4 .998t/ hm2 ,其中植被部分为 1 54.2 89t/ hm2 ,土壤为 89.1 2 8t/ hm2 ,地表凋落物层为 1 .581 t/ hm2。黄果厚壳桂、鼎湖钓樟群落植被碳总贮量为 84.1 51 t/ hm2。在两林分植被碳总贮量中 ,乔木层分别占了97.47% (锥栗、黄果厚壳桂群落 )和 98.0 4 % (黄果厚壳桂、鼎湖钓樟群落 ) ,而在乔木层碳总贮量中 ,干器官则分别占 47.93% (锥栗、黄果厚壳桂群落 )和 44.66% (黄果厚壳桂、鼎湖钓樟群落 )。锥栗、黄果厚壳桂群落植被碳年积累量为 3.1 4 9t/ (hm2· a) ,黄果厚壳桂、鼎湖钓樟群落植被碳年积累量则为 3.42 5 t/ (hm2· a)。     

Cytoskeletal scaffolding proteins interact with Lynch‐Syndrome associated mismatch repair protein MLH1

The involvement of MLH1 in several mismatch repair‐independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two‐hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the “General Repository for Interaction Datasets” highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin β 4 and Actin γ, Cathepsin B and Annexin A2 as well as Spectrin α and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1‐deficient and ‐proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1–Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.     

林下层植物在退化马尾松林恢复初期养分循环中的作用

以鼎湖山退化马尾松 (Pinusmassoniana)林恢复过程中林下层植物凋落物、分解和养分动态为对象 ,研究了林下层植物在退化马尾松林恢复初期养分循环中的作用。结果表明 ,林下层年凋落物量除在第 5年有所下降外均随时间逐年上升 ,但其增加速率随年份不同而异 ,总平均年增长速率为 3 8%。第 4年凋落物量为 0 .2 0 t· hm- 2 · a- 1,第 1 1年为 1 .1 7t·hm- 2·a- 1。凋落物养分元素平均浓度为 (% ) :N0 .95 ,P0 .0 4,K0 .5 7,Ca0 .1 3和 Mg0 .0 8,基本上以夏季和秋季最高冬春交替月份最低。第 1 1年凋落物各元素养分归还量为 (kg· hm- 2·a- 1) :N1 1 .1 0 ,P0 .47,K6.65 ,Ca1 .48和 Mg 0 .91。凋落物在分解过程中失重率呈直线模型变化 ,第 1年的分解速率为 3 1 % ,至试验结束时凋落物的残存量占起始量的 66%。在凋落物分解过程中 ,N和 P浓度随时间逐渐上升 ,但 N增加的速度较 P快 ,其余元素浓度均下降 ,但 K下降的速度最快。在凋落物分解过程中 ,N是唯一表现残留量呈先上升然后下降变化的元素。P的残留量变化与凋落物的失重率变化几乎一致。各元素在分解试验结束时残留量占起始量的百分比分别为 :N 90 % ,P 67% ,K 9% ,Ca 3 0 %和Mg 1 4%。可见 ,林下层凋落物在退化马尾松林恢复初期碳及其它营养元素循     

A strain of <Emphasis Type="Italic">Meyerozyma guilliermondii</Emphasis> isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.