人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

Structure,dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK

The enzyme m¹A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNA^Leu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.     

Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.     

Breeding bird communities of second‐rotation plantations at different stages of the forest cycle

Capsule Early stages of the plantation forest cycle have distinct bird communities and bird density was significantly higher in the second rotation than in the first for a given age class.

Aims To characterize the bird communities in Irish second-rotation plantations and to compare them with those of first-rotation plantations.

Methods Point counts were used to survey 20 plantation forests in four age classes (Pre-thicket; Thicket; Mid‐rotation; and Mature) in the breeding season of 2007. distance software was used to generate bird densities. Ordination, indicator species analysis, and glm were used to analyse the bird communities.

Results Bird communities of Pre‐thicket and, to a lesser extent, Thicket age classes were distinct from those of more mature forests. Bird communities of Mid‐rotation and Mature age classes were indistinguishable from each other and were therefore combined into a single age class (Closed canopy). Pre‐thicket held significantly lower total bird density, but significantly higher migrant bird density, than this Closed canopy age class. Bird density was significantly higher in the second rotation in all age classes except for Pre‐thicket, but migrant density was significantly higher in Pre‐thicket in the second rotation. There was no difference in species richness between the first and second rotation.

Conclusions Differences between rotations are probably due to changes in vegetation structure, and the increase in second‐rotation forests in Ireland is likely to be a positive development for bird communities. Especially encouraging is the higher migrant bird density in second‐rotation Pre‐thicket, as some of these species are in decline throughout Europe. However, the largest differences in population density between rotations were exhibited by common species and such species will likely benefit most from future increases in the area of second‐rotation plantation forests.     

Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61

Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization.     

The influence of a native tree species mix component on bird communities in non‐native coniferous plantations in Ireland

Capsule Norway Spruce plantations with Scots Pine as a secondary tree species had higher bird densities than pure Norway Spruce. Shrub cover was the most important structural variable, influencing bird density, species richness and Simpson’s diversity.

Aims To investigate whether incorporating a native tree component into non‐native coniferous plantations had any effect on bird communities or vegetation structure.

Methods Birds were surveyed in plantations of Norway Spruce mixed with Oak and Scots Pine, each paired with a plantation of pure Norway Spruce. distance was used to generate bird densities. Bird density, species richness and Simpson’s diversity were compared between each mix type and pure Norway Spruce. glms were used to investigate relationships between structural components of plantations and bird data.

Results Bird communities of mixed plantations differed only slightly in their composition from pure Norway Spruce. Bird density was significantly higher in Scots Pine mixes than in Oak mixes or pure Norway Spruce. Neither species richness nor Simpson’s diversity differed significantly between the plantation types. Some vegetation components differed between the plantations and shrub cover was positively associated with bird density, species richness and Simpson’s diversity. The presence of rides also increased bird density.

Conclusions There is a positive effect on bird communities of including a native tree species in non‐native coniferous plantations, but the magnitude of the effect is small. The influence of shrub cover on birds suggests that forest management may play an important role in determining the utility of plantations for birds. We recommend the establishment of mixed tree species plantations where possible, although, in the case of Oak mixes, the Norway Spruce appeared to suppress growth of the Oak and thus may be restricting its effect on birds. Changes in management, such as planting Oaks in clumps or heavier thinning of the coniferous component, could address this problem.     

An introductory biology lab that uses enzyme histochemistry to teach students about skeletal muscle fiber types

One important goal of introductory biology laboratory experiences is to engage students directly in all steps in the process of scientific discovery. Even when laboratory experiences are built on principles discussed in the classroom, students often do not adequately apply this background to interpretation of results they obtain in lab. This disconnect has been described at the level of medical education (4), so it should not be surprising that educators have struggled with this same phenomenon at the undergraduate level. We describe a new introductory biology lab that challenges students to make these connections. The lab utilizes enzyme histochemistry and morphological observations to draw conclusions about the composition of functionally different types of muscle fibers present in skeletal muscle. We report that students were not only successful at making these observations on a specific skeletal muscle, the gastrocnemius of the frog Rana pipiens, but that they were able to connect their results to the principles of fiber type differences that exist in skeletal muscles in all vertebrates.     

Partial characterization and ontogeny of renal cytosolic glucocorticoid receptors in mouse kidney

The glucocorticoid receptor (GR) was partially characterized in mouse renal cytosol. A sensitive and reproducible [3H]dexamethasone binding assay suitable for use with small quantities of cytosolic protein, was developed. Studies defined the optimal equilibrium binding conditions, metabolism of [3H]dexamethasone in adult renal cytosol, specificity of binding of the GR, and molecular weight of the GR-[3H]dexamethasone complex by gel filtration chromatography. The assay was subsequently used to measure the renal GR during different stages of foetal and postnatal development, as well as in glomerular and renal tubular preparations from adult mice. An almost linear increase in GR occurred from day 13 to day 18 of gestation with levels rising from 100 to 201 fmol/mg cytosol protein; this was followed by a sharp rise in receptor concentration just after birth to 343 fmol/mg cytosol protein. Adult levels, 410-433 fmol/mg cytosol protein, were reached by 2 weeks after birth. The equilibrium dissociation constants (Kd) of the [3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8-11.8 nM; mean +/- SD = 6.5 +/- 2.9 nM). Specific binding was assessed to be 3- to 5-fold greater in tubular than in glomerular preparations. These data on the localization and ontogeny of GR during murine metanephric development provide a basis for study of glucocorticoid-mediated effects on various models of congenital and acquired renal disease.     

Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1-2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.