Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.     

Observations on egg-laying behaviour of the banana weevil,Cosmopolites sordidus (Germar)

Banana weevil females laid on an average 2.7 eggs/week in rhizome material and 0.7 eggs/week in pseudostem material in the laboratory. At extremely high weevil population densities the egg-laying activity declined. Under controlled field conditions 0.7 eggs/week were laid in banana suckers and 1.3 eggs/week in stumps of harvested suckers. 25% of the weevil stages found in suckers in the field were eggs of which 78% were laid in the rhizome and 22% in the pseudostem base. The majority of eggs was deposited in the crown area of the rhizome followed by the remaining surface area of the rhizome, the walls of abandoned larval tunnels in rhizome and pseudostem and the leaf sheaths. 58% of the eggs found were considered accessible to egg predators.     

Synthetic glycosylation of peptides using unprotected saccharide β-glycosylamines

Glycopeptides can be valuable tools in determining the influence of carbohydrate moieties on the intrinsic properties of glycoproteins. However, glycopeptides of sufficient quantity and purity are as yet not readily available from biological sources. The chemical coupling of a -glycosylamino group of an unprotected carbohydrate with an activated aspartic acid residue of an unprotected peptide is a simple method for synthesizing asparagine-linked glycopeptides. In this report we demonstrate that the use of this method is not restricted to -glycosylamines of simple monosaccharides or short aspartic acid-containing pentapeptides. This is illustrated by the syntheses of several glycopentapeptides containingN,N-diacetylchitobiose, a glutamine-linked glycopentapeptide containing a biantennary complex oligosaccharide, and glycosylated variants of two analogs of a polypeptide hormone, atriopeptin, containingN,N-diacetylchitobiose.Abbreviations Ac acetyl - Bzl benzyl - DMF dimethylformamide - Fmoc 9-fluorenylmethoxycarbonyl - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - HBTU O-benzotriazol-1-yl-N,N,N,N-tetramethyluroniumhexa-fluorophosphate - HOBt 1-hydroxybenzotriazole - Man mannose - m/z mass/charge - NMR nuclear magnetic resonance - Xyl xylose - Z benzyloxycarbonyl; unless otherwise specified, amino acids are abbreviated using their one-letter codes.     

Coevolution-Guided Mapping of the Type VI Secretion Membrane Complex-Baseplate Interface

The type VI secretion system (T6SS) is a multiprotein weapon evolved by Gram-negative bacteria to deliver effectors into eukaryotic cells or bacterial rivals. The T6SS uses a contractile mechanism to propel an effector-loaded needle into its target. The contractile tail is built on an assembly platform, the baseplate, which is anchored to a membrane complex. Baseplate-membrane complex interactions are mainly mediated by contacts between the C-terminal domain of the TssK baseplate component and the cytoplasmic domain of the TssL inner membrane protein. Currently, the structural details of this interaction are unknown due to the marginal stability of the TssK-TssL complex. Here we conducted a mutagenesis study based on putative TssK-TssL contact pairs identified by co-evolution analyses. We then evaluated the impact of these mutations on T6SS activity, TssK-TssL interaction and sheath assembly and dynamics in enteroaggregative Escherichia coli. Finally, we probed the TssK-TssL interface by disulfide cross-linking, allowing to propose a model for the baseplate-membrane complex interface.     

中国旧石器原料开发策略的研究进展

石料开发与获取是石器生产操作链的重要环节,也是史前人地关系研究的重要内容,能够反映史前人类的资源开发策略、环境认知与适应能力、移动模式和交流贸易网络等。相比于旧大陆西侧,东亚地区的燧石和黑曜岩等优质石料产地相对稀缺,中国许多旧石器考古遗址的石器原料主要来自遗址周边质量欠佳的脉石英、石英岩和一般燧石等,而相关的旧石器时代石料开发策略系统研究也相对较少。本文对中国目前已开展的旧石器时代石料开发策略研究进行系统梳理分析和总结发现,从旧石器时代早期到晚期末段,中国境内史前人类的石料开发策略存在明显的时空变化,主要体现在石料类型组成、产地选取和开发方式三个方面,可能主要受到史前人类流动性变化、石器技术演变和环境变化等因素的共同影响。综合而言,开展旧石器时代遗址的石料开发策略研究,对于理解中国境内史前人类的行为模式和交流迁徙等有重要意义。     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

Effects of phosphorylation of P-glycoprotein on multidrug resistance

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.     

Habitat selection by grayling—I. Spawning habitats

Grayling spawning sites were investigated in two French rivers, the Pollon (1 year) and the Suran (2 years) and described by current velocity, water depth, and substrate composition, completed by an assessment of bottom shear stress with FST-hemispheres. A comparison was made between used and available habitats, the latter being characterized by random sampling of 300 m long (Pollon) and 510 m long (Suran) river sections, both including three riffle/pool sequences. Mean velocities observed on spawning sites did not differ significantly between rivers or years (overall mean 48–9 cm s^-1 S.D. = 11.9, range 25.8–91.7 cm s^-1, n =150). Most water depths ranged from 10 to 40 cm in both rivers, but mean depths were significantly different ( P <0.05). Substratum of spawning grounds was dominated by gravel and pebbles (2–64 mm) in both rivers. Most spawning sites (99%) were characterized by a narrow range of hemispheres (nos 9–13), i.e. a range of shear stress of about 5–16 dyn m^-2. In the Pollon, spawners between spawning acts were found in a resting pool located immediately downstream from the spawning area and characterized by slow-flowing water (mainly <20 cm s^-1) and great depth (mainly >60 cm), with cover provided by overhanging branches and tree roots.     

Evaluation of several methods for estimating phylogenetic trees when substitution rates differ over nucleotide sites

Several maximum likelihood and distance matrix methods for estimating phylogenetic trees from homologous DNA sequences were compared when substitution rates at sites were assumed to follow a gamma distribution. Computer simulations were performed to estimate the probabilities that various tree estimation methods recover the true tree topology. The case of four species was considered, and a few combinations of parameters were examined. Attention was applied to discriminating among different sources of error in tree reconstruction, i.e., the inconsistency of the tree estimation method, the sampling error in the estimated tree due to limited sequence length, and the sampling error in the estimated probability due to the number of simulations being limited. Compared to the least squares method based on pairwise distance estimates, the joint likelihood analysis is found to be more robust when rate variation over sites is present but ignored and an assumption is thus violated. With limited data, the likelihood method has a much higher probability of recovering the true tree and is therefore more efficient than the least squares method. The concept of statistical consistency of a tree estimation method and its implications were explored, and it is suggested that, while the efficiency (or sampling error) of a tree estimation method is a very important property, statistical consistency of the method over a wide range of, if not all, parameter values is prerequisite.