Towards a molecular understanding of cytokinesis

In this review, we focus on recent discoveries regarding the molecular basis of cleavage furrow positioning and contractile ring assembly and contraction during cytokinesis. However, some of these mechanisms might have different degrees of importance in different organisms. This synthesis attempts to uncover common themes and to reveal potential relationships that might contribute to the biochemical and mechanical aspects of cytokinesis. Because the information about cytokinesis is still fairly rudimentary, our goal is not to present a definitive model but to present testable hypotheses that might lead to a better mechanistic understanding of the process.     

Towards understanding selenocysteine incorporation into bacterial proteins

In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high-resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.     

DNA segregation by bacterial actin homologs

The bacterial actin homolog ParM catalyzes segregation of plasmid DNA in E. coli. Recent studies now suggest a model in which ParM forms actin-like filaments between two plasmid molecules, thereby providing the driving force for plasmid DNA separation.     

Towards understanding molecular modes of probiotic action

The possibility that certain microorganisms might be beneficial to human health is highlighted by the numerous consumer products containing probiotic bacteria. Probiotics are typically administered in food that, following entry into the gastro-intestinal tract, results in measurable health-promoting effects. Although there is a growing list of health benefits provided by the consumption of probiotics, their precise mechanisms of action remain largely unknown. Recent molecular- and genomics-based studies are starting to provide insight into the ways probiotic bacteria sense and adapt to the gastro-intestinal tract environment. Complementary approaches using host cell in vitro systems together with animal models and human volunteers are revealing specific intestinal cell responses to probiotics. These studies should ultimately disclose the molecular mechanisms and pinpoint the bacterial and host effector molecules and pathways by which probiotics are able to modulate human health.     

Towards a molecular understanding of titin.

Titin is at present the largest known protein (M(r) 3000 kDa) and its expression is restricted to vertebrate striated muscle. Single molecules span from M- to Z-lines and therefore over 1 micron. We have isolated cDNAs encoding five distant titin A-band epitopes, extended their sequences and determined 30 kb (1000 kDa) of the primary structure of titin. Sequences near the M-line encode a kinase domain and are closely related to the C-terminus of twitchin from Caenorhabditis elegans. This suggests that the function of this region in the titin/twitchin family is conserved throughout the animal kingdom. All other A-band sequences consist of 100 amino acid (aa) repeats predicting immunoglobulin-C2 and fibronectin type III globular domains. These domains are arranged into highly ordered 11 domain super-repeat patterns likely to match the myosin helix repeat in the thick filament. Expressed titin fragments bind to the LMM part of myosin and C-protein. Binding strength increases with the number of domains involved, indicating a cumulative effect of multiple binding sites for myosin along the titin molecule. We conclude that A-band titin is likely to be involved in the ordered assembly of the vertebrate thick filament.     

Towards a molecular understanding of Drosophila hearing

The Drosophila auditory system is presented as a powerful new genetic model system for understanding the molecular aspects of development and physiology of hearing organs. The fly's ear resides in the antenna, with Johnston's organ serving as the mechanoreceptor. New approaches using electrophysiology and laser vibrometry have provided useful tools to apply to the study of mutations that disrupt hearing. The fundamental developmental processes that generate the peripheral nervous system are fairly well understood, although specific variations of these processes for chordotonal organs (CHO) and especially for Johnston's organ require more scrutiny. In contrast, even the fundamental physiologic workings of mechanosensitive systems are still poorly understood, but rapid recent progress is beginning to shed light. The identification and analysis of mutations that affect auditory function are summarized here, and prospects for the role of the Drosophila auditory system in understanding both insect and vertebrate hearing are discussed.     

Towards the molecular understanding of glycogen elongation by amylosucrase

Amylosucrase from Neisseria polysaccharea (AS) is a transglucosidase from the glycoside-hydrolase family 13 that catalyzes the synthesis of an amylose-like polymer from sucrose, without any primer. Its affinity towards glycogen is particularly noteworthy since glycogen is the best D-glucosyl unit acceptor and the most efficient activator (98-fold k(cat) increase) known for this enzyme. Glycogen-enzyme interactions were modeled starting from the crystallographic AS: maltoheptaose complex, where two key oligosaccharide binding sites, OB1 and OB2, were identified. Two maltoheptaose molecules were connected by an alpha-1,6 branch by molecular modeling to mimic a glycogen branching. Among the various docking positions obtained, four models were chosen based on geometry and energy criteria. Robotics calculations enabled us to describe a back and forth motion of a hairpin loop of the AS specific B'-domain, a movement that assists the elongation of glycogen branches. Modeling data combined with site-directed mutagenesis experiments revealed that the OB2 surface site provides an anchoring platform at the enzyme surface to capture the polymer and direct the branches towards the OB1 acceptor site for elongation. On the basis of the data obtained, a semiprocessive glycogen elongation mechanism can be proposed.     

动物源细菌耐药性研究现状与对策

目前我国动物源细菌耐药现象十分普遍,多重耐药甚至泛耐药的菌株不断出现,给公共卫生和食品安全造成了重大威胁。文中从我国动物源耐药性研究的主要问题、细菌耐药性形成和传播机制以及耐药性防控策略等几个方面进行综述,以期为耐药性的研究和防控提供参考。     

Towards an understanding of the genetics of bacterial metal resistance.

Many bacteria show great promise for use in metal recovery. However, the genetics of metal-leaching, accumulation-resistance, and oxidation/reduction mechanisms of these bacteria is still an area of research in its infancy. The introduction of such genes into bacteria of economic importance would have application in biomining and environmental bioremediation.     

Towards an understanding of the molecular basis of effective RNAi against a global insect pest,the whitefly Bemisia tabaci

In planta RNAi against essential insect genes offers a promising route to control insect crop pests, but is constrained for many insect groups, notably phloem sap-feeding hemipterans, by poor RNAi efficacy. This study conducted on the phloem-feeding whitefly Bemisia tabaci reared on tomato plants investigated the causes of low RNAi efficacy and routes to ameliorate the problem. Experiments using tomato transgenic lines containing ds-GFP (green fluorescent protein) revealed that full-length dsRNA is phloem-mobile, ingested by the insects, and degraded in the insect. We identified B. tabaci homologs of nuclease genes (dsRNases) in other insects that degrade dsRNA, and demonstrated that degradation of ds-GFP in B. tabaci is suppressed by administration of dsRNA against these genes. dsRNA against the nuclease genes was co-administered with dsRNA against two insect genes, an aquaporin AQP1 and sucrase SUC1, that are predicted to protect B. tabaci against osmotic collapse. When dsRNA constructs for AQP1, SUC1, dsRNase1 and dsRNase2 were stacked, insect mortality was significantly elevated to 50% over 6 days on artificial diets. This effect was accompanied by significant reduction in gene expression of the target genes in surviving diet-fed insects. This study offers proof-of-principle that the efficacy of RNAi against insect pests can be enhanced by using dsRNA to suppress the activity of RNAi-suppressing nuclease genes, especially where multiple genes with related physiological function but different molecular function are targeted.     

Towards understanding how molecular networks evolve in plants

Residing beneath the phenotypic landscape of a plant are intricate and dynamic networks of genes and proteins. As evolution operates on phenotypes, we expect its forces to shape somehow these underlying molecular networks. In this review, we discuss progress being made to elucidate the nature of these forces and their impact on the composition and structure of molecular networks. We also outline current limitations and open questions facing the broader field of plant network analysis.     

Towards an understanding of the molecular basis of plants K+ transport: Characterization of cloned K+ transport cDNAs

Recently, two K⁺-transport cDNAs, KAT1 and AKT1, were cloned in Arabidopsis thaliana. These cDNAs had structural similarities to K⁺ channel genes in animals, and also conferred the ability for growth on micromolar levels of K⁺ when expressed in K⁺ transport-defective yeast mutants. In this study, we examined the possibility that KAT1 encodes the high-affinity K⁺ transport system that has been previously characterized in plant roots, by studying the concentration-dependent kinetics of K⁺ transport for KAT1 expressed in Xenopus oocytes and Saccharomyces cerevisiae. In both organisms, the K⁺ transport system encoded by KAT1 yielded Michaelis-Menten kinetics with a high K_m for K⁺ (35 mM in oocytes, 0.6 mM in yeast cells). Furthermore, Northern analysis indicated that KAT1 is expressed primarily in the Arabidopsis shoot. These results strongly suggest that the system encoded by KAT1 is not a root high-affinity K⁺ transporter.     

多巴胺重摄取的分子机制

多巴胺转运体(Dopamine transporter,DAT)位于多巴胺能神经元表面,主要负责将细胞外的多巴胺重摄取至多巴胺能神经元内,控制细胞外多巴胺的浓度,进而影响多巴胺的信号强度和时长。多巴胺转运体与注意力缺陷多动症、抑郁、成瘾等中枢神经系统的功能异常相关。多巴胺转运体的重摄取功能受多种因素的调节,包括底物的浓度、自身位点的翻译后修饰、细胞内蛋白激酶的活性、细胞外的调节信号等。本文就近年来DAT的分子调节机制以及在脑疾病发病机制中作用的研究进展做一综述。     

Towards a molecular understanding of polycythemia rubra vera.

Polycythemia rubra vera (PV) is one of four diseases collectively called the myeloproliferative disorders (MPDs). Each disorder leads to an increased production of one or several hematopoietic cell lineages. MPDs arise from acquired mutations in a pluripotent hematopoietic stem cell. However, the molecular mechanisms leading to the development of these diseases are poorly understood. This review will summarize and evaluate recent advances in our understanding of one particular MPD, PV.     

Preeclampsia: current understanding of the molecular basis of vascular dysfunction

Preeclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria occurring after the 20th week of gestation. Delivery of the placenta results in resolution of the condition, implicating the placenta as a central culprit in the pathogenesis of preeclampsia. In preeclampsia, an inadequate placental trophoblast invasion of the maternal uterine spiral arteries results in poor placental perfusion, leading to placental ischaemia. This could result in release of factors into the maternal circulation that cause widespread activation or dysfunction of the maternal endothelium. Factors in the maternal circulation might induce oxidative stress and/or elicit an inflammatory response in the maternal endothelium, resulting in the altered expression of several genes involved in the regulation of vascular tone. This review addresses the potential circulating factors and the molecular mechanisms involved in the alteration of vascular function that occurs in preeclampsia.     

Towards an understanding of the structural basis for insect olfaction by odorant receptors

Insects have co-opted a unique family of seven transmembrane proteins for odour sensing. Odorant receptors are believed to have evolved from gustatory receptors somewhere at the base of the Hexapoda and have expanded substantially to become the dominant class of odour recognition elements within the Insecta. These odorant receptors comprise an obligate co-receptor, Orco, and one of a family of highly divergent odorant “tuning” receptors. The two subunits are thought to come together at some as-yet unknown stoichiometry to form a functional complex that is capable of both ionotropic and metabotropic signalling. While there are still no 3D structures for these proteins, site-directed mutagenesis, resonance energy transfer, and structural modelling efforts, all mainly on Drosophila odorant receptors, are beginning to inform hypotheses of their structures and how such complexes function in odour detection. Some of the loops, especially the second extracellular loop that has been suggested to form a lid over the binding pocket, and the extracellular regions of some transmembrane helices, especially the third and to a less extent the sixth and seventh, have been implicated in ligand recognition in tuning receptors. The possible interaction between Orco and tuning receptor subunits through the final intracellular loop and the adjacent transmembrane helices is thought to be important for transducing ligand binding into receptor activation. Potential phosphorylation sites and a calmodulin binding site in the second intracellular loop of Orco are also thought to be involved in regulating channel gating. A number of new methods have recently been developed to express and purify insect odorant receptor subunits in recombinant expression systems. These approaches are enabling high throughput screening of receptors for agonists and antagonists in cell-based formats, as well as producing protein for the application of biophysical methods to resolve the 3D structure of the subunits and their complexes.