Spatial organization of the synthesis of cytoskeletal proteins

The cytoskeleton of most cells is complex and spatially diverse. The mRNAs for some cytoskeletal proteins are localized, suggesting that synthesis of these proteins may occur at sites appropriate for function or assembly. mRNA concentrations were first observed for several oocyte and embryonic mRNAs. Some insight has been gained into the mechanisms that help to position these mRNAs. More surprising to some, many cytoskeletal mRNAs are also localized. Among them are mRNAs for actin, tubulin, intermediate filaments, and a variety of associated proteins. Different mRNAs in the same cell can be located in different places; the same mRNA can be located in different places; the same mRNA can be located differently at different times of development. For example, we observed vimentin mRNA in developing chicken muscle cultures by fluorescent in situ hybridization. We found that vimentin mRNA takes on a variety of positions during myogenesis, ending up located with its cognate protein at costameres. This last pattern is significant because it is too finely structured to have a function in the soluble phase and probably reflects contranslational assembly of this particular protein. Analogies can be made between oocyte or embryonic positions (animal/vegetal poles, oocyte cortex, and interior) and somatic cell positions (anterior/posterior and cell cortex/cell center). These analogies may point to conserved mechanisms for moving and retaining mRNA. Localization of cytoskeletal synthesis, through the mRNA or by other means, may prove as important for assembling and maintaining differentiated cytoskeletal structures and somatic cells as mRNA location is for organizing the embryo. Mechanisms that permit mRNA localization are likely to be conserved.     

信号分子在生物脱氮中的作用及检测方法

生物脱氮是由微生物主导的地球氮循环中的重要环节之一,主要包括硝化、反硝化和厌氧氨氧化(anaerobic ammonium oxidation,anammox)等过程。在微生物联合作用下,污水中的有机氮及氨氮经一系列作用转化为氮气,这种经济高效、环境友好的处理工艺在世界范围内得到广泛应用。群体感应(quorum sensing,QS)以信号分子为媒介通过改变菌群密度和周围环境变化来调节微生物的各种行为。大量的研究已证实调控QS信号分子在生物脱氮中具有应用潜力。本文介绍了各种信号分子类型,从基因组学、实际应用等方面综述了各类信号分子以及检测方法,同时针对酰基高丝氨酸内酯(acyl homoserine lactones,AHLs)类信号分子在生物脱氮中的作用进行详细介绍。然而不足之处在于信号分子研究只是停留在实验室阶段,仅仅研究了单一信号分子对生物脱氮的影响。未来可将信号分子应用于实际污水,研究多种信号分子共同作用以及多种微生物之间的QS现象。     

基于果蝇口腔感染的肠道训练免疫模型研究（英文）

目的 利用果蝇作为遗传工具从个体和分子层面研究果蝇的训练免疫效应,并为后续深入研究其分子机制提供依据。方法 首先构建无菌果蝇模型,在此基础上构建果蝇成虫及跨发育阶段训练免疫模型,用两种革兰氏阴性菌——胡萝卜软腐欧文氏菌（Erwinia carotovora carotovora 15）及铜绿假单胞菌（Pseudomonas aeruginosa）分别经口腔感染果蝇。在第一次感染完全消退后进行再次感染,然后通过比较果蝇在两个感染阶段的存活率和细菌量来衡量训练免疫的潜在效果。通过实时荧光定量PCR检测相应先天免疫相关基因的表达水平,研究革兰氏阴性菌对免疫缺陷（IMD）通路的诱导作用。结果 果蝇成虫及幼虫初次感染均可提高二次感染后的生存率、细菌清除效率及死亡时能承受的最高细菌负荷;二次感染的果蝇中,IMD通路中免疫反应基因的基础表达比未感染的高,这提供了获得感染抗性的分子基础;果蝇的免疫反应主要发生在中肠,二次免疫比初次免疫的效应更迅速且剧烈;二次免疫的果蝇中,肠道干细胞的数量显著多于初次感染。结论 果蝇肠道中强大的训练免疫可由同源或异源革兰氏阴性菌口腔感染引发,且免疫记忆可在整个发育阶段持...     

依达拉奉右莰醇联合丁苯酞治疗对老年大动脉粥样硬化型脑卒中患者血清PTX3、lp-PLA2以及MES的影响

摘要 目的：探讨依达拉奉右莰醇联合丁苯酞对老年大动脉粥样硬化（LAA）型脑卒中患者血清五聚素3（PTX3）、脂蛋白相关磷脂酶A2（Lp-PLA2）以及微栓子信号（MES）的影响。方法：回顾性选取2021年9月-2022年9月在本院收治的120例老年LAA型脑卒中患者,根据其不同治疗方案分为对照组（56例）,采用依达拉奉右莰醇单独治疗,和观察组（64例）,采用依达拉奉右莰醇联合丁苯酞治疗。观察两组患者的临床疗效;对比两组患者治疗前后神经功能[脑卒中量表（NIHSS）及中枢神经特异性蛋白（S-100β）、神经元特异性烯醇化酶（NSE）]状态、炎症因子[血清五聚素3（PTX3）、脂蛋白相关磷脂酶A2（Lp-PLA2）]水平及脑血流微栓子信号（MES）阳性率变化。记录不良反应发生情况。结果：观察组患者治疗有效率为95.31%,高于对照组的78.57%（χ²=7.653 ,P=0.006）;治疗后,观察组患者NIHSS评分、S-100β及NSE水平低于对照组（P＜0.05）;观察组PTX3、Lp-PLA2水平及MES阳性率低于对照组（P<0.05）。观察组与对照组总不良反应发生率比较无差异（6.25 % vs 5.36 %）（Fisher=1.000）。结论：依达拉奉右莰醇联合丁苯酞可有效提高临床疗效,促进老年LAA型患者神经功能恢复,降低PTX3、lp-PLA2水平及MES阳性率,控制病情发展,且安全性较好。     

MONOCLONAL ANTIBODIES AS MOLECULAR MARKERS FOR THE INTRACELLULAR AND CELL WALL DISTRIBUTION OF CARRAGEENAN EPITOPES IN KAPPAPHYCUS (RHODOPHYTA) DURING TISSUE DEVELOPMENT1

Carrageenan, the major cell wall carbohydrate of certain red algae, is variable in structure and gelling properties. Sequence types include gelling (kappa and iota) and nongelling (lambda) types in addition to precursors, often in hybrid molecules containing more than one precursor and/or sequence type. Molecular markers to subunits were needed to study carrageenan synthesis, cell wall organization, and the relationship between structure and function. Monoclonal antibodies were produced to carrageenan, and their specificities were determined by competitive enzyme immunoassay. Antibodies were identified with specificities related to kappa, iota, and lambda carrageenan. The patterns of immunofluorescence localization on Kappaphycus alvarezii = Eucheuma alvarezii var. tambalang (Doty) sections were distinctive for each antibody. The antibody to a kappa-related epitope labeled mature tissue strongly; antibodies to an iota-related epitope and a lambda-related epitope labeled weakly, consistent with the kappa-enriched carrageenan produced by this alga. Kappa-related epitopes were distributed throughout the wall and matrix, whereas iota-related epitopes were concentrated in the middle lamella. Lambda-related epitopes were localized primarily at the plant cuticle where kappa and iota antigens were lacking. An antibody appeared to be specific for a precursor of the gelling subunits because it showed maximal wall and intracellular labeling at the youngest developmental stage. All antibodies labeled intracellular inclusions in the transition zone between the epidermis and medulla during the development of medullary cells from the peripheral meristem in young branches. The results demonstrate the intracellular synthesis of epitopes related to all major carrageenan subunits and their differential extracellular distribution.     

Strategies for microarray analysis of limiting amounts of RNA.

One of the critical limitations of current microarray technologies for use in expression analyses is the relatively large amount of input RNA required to generate labelled cDNA populations for array analysis. In situations where RNA is limiting, the options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell.     

Interaction of synthetic peptide corresponding to signal sequence of glucitol permease of Escherichia Coli and its analogue with liposomes

The N-terminal signal sequence of glucitol pcrmease of Escherichia Coli (Gut22) and its analogue (Gut22Ana) were synthesized. The analogue had a Pro residue substituting for the His at the 7th position of Gut22 and a Val residue substituting for the Glu at the 10th position. The intrinsic fluorescence emission spectra indicated that the binding of Gut22 with lipid bilayer was much stronger than that of Gut22Ana. The leakage experiments with calcein-loaded liposomes showed that Gut22 strongly perturbed lipid bilayers while Gut22Ana did not. The apparent partition constant of Gut22 for partitioning into phosphatidylserine/phosphatidylcholine bilayers was measured; the effect of membrane potential on the interaction of Gut22 with lipid bilayers was studied and the conformation changes of Gut22 and Gut22Ana upon interacting with liposomes were studied by the method of circular dichroism analysis.     

Secondary structure of β-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from Hα, Cα, Cβ and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

Summary Nearly complete backbone ¹H, ¹⁵N and ¹³C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although ¹⁵N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins. Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D ¹H–¹⁵N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and ³J_HN data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed.     

环化二磷酸腺苷核糖及其钙动员的功能

环化二磷酸腺苷核糖（cyclic ADP-ribose,cADPR）是烟酰胺腺嘌呤二核苷酸(NAD⁺)的代谢产物,是新近发现的一种细胞内第二信使．在许多哺乳类和无脊椎动物细胞中,cADPR能引起胞内钙库释放钙离子,其可能机制是：cADPR受体结合cADPR,通过Ryanodine受体或类Ryanodine受体介导的钙通道使cADPR敏感的钙库释放钙离子,此外,一条由一氧化氮（NO）、环化鸟苷酸（cGMP）和cADPR组成的细胞内信号转导途径可能存在于许多细胞中．     

Reconstitution of a pentameric complex of dimeric transforming growth factor beta ligand and a type I,II, III receptor in baculoviral-infected insect cells

Summary Two transmembrane serine-threonine kinases (type I and II receptors), a membrane-anchored proteoglycan (type III), and a homodimeric ligand participate in the transforming growth factor beta type on (TGFβ1) signal transduction complex. The expression of recombinant receptors in insect cells co-infected with up to three recombinant baculoviruses was employed to study interactions among the ectodomains of the three types of receptors and the TGFβ1 ligand in absence of uncontrollable extrinsic factors in mammalian cells. Multi-subunit complexes were assembled in intact cells and purified on glutathione-conjugated beads for analysis by tagging one of the subunits with glutathione S-transferase (GST). Intrinsic ligand-independent interactions were observed among receptor subunits as follows: type III–III type I–I, type III-I, and type II-I. The homeotypic complex of type II–II receptors and the heterotypic type III-II interaction was ligand dependent. The type I, but not the type III, subunit displaced about 50% of the type II component in either ligand-dependent homomeric type II-type II complexes or heteromeric type III-type II complexes to form type II-I or type III-II-I oligomers, respectively. The type II subunit displaced type I subunits in oligomers of the type I subunit. Specificity of type I receptors may result from differential affinity for the type II receptor rather than specificity for ligand. A monomeric subunit of the TGFβ1 ligand bound concurrently to type III and type II or type III and type I receptors, but failed to concurrently bind to the type II and type I subunits. The binding of TGFβ1 to the type I kinase subunit appears to require an intact disulfide-linked ligand dimer in the absence of a type III subunit. The combined results suggest a pentameric TGFβ signal transduction complex in which one unit each of the type III, type II, and type I components is assembled around the two subunits of the dimeric TGFβ1 ligand. An immobilized GST-tagged subunit of the receptor complex was utilized to assemble multi-subunit complexesin vitro and to study the phosphorylation events among subunits in the absence of extrinsic cell-derived kinases. The results revealed that (a) a low level of ligand-independent autophosphorylation occurs in the type I kinase; (b) a high level of autophosphorylation occurs in the type II kinase; (c) both the type III and type I subunits aretrans-phosphorylated by the type II subunit; and (d) the presence of both type I and II kinases complexed with the type III subunit and dimeric TGFβ1 ligand in a pentameric complex causes maximum phosphorylation of all three receptor subunits.