Use of bicuculline, a GABA antagonist, as a template for the development of a new class of ligands showing positive allosteric modulation of the GABA(A) receptor

Analogues of bicuculline devoid of the benzo ring fused to the lactone moiety were prepared by reacting 2-(tert-butyl-dimethylsiloxy)furans with 3,4-dihydroisoquinolinium salts. Some of these compounds (e.g., ROD185, 8) acted as modulators of the GABAA receptor, displacing ligands of the benzodiazepine binding site. They also strongly stimulated GABA currents mediated by recombinant GABA(A) receptors expressed in Xenopus oocytes.     

Area per lipid and acyl length distributions in fluid phosphatidylcholines determined by (2)H NMR spectroscopy

Deuterium ((2)H) NMR spectroscopy provides detailed information regarding the structural fluctuations of lipid bilayers, including both the equilibrium properties and dynamics. Experimental (2)H NMR measurements for the homologous series of 1, 2-diacyl-sn-glycero-3-phosphocholines with perdeuterated saturated chains (from C12:0 to C18:0) have been performed on randomly oriented, fully hydrated multilamellar samples. For each lipid, the C-D bond order parameters have been calculated from de-Paked (2)H NMR spectra as a function of temperature. The experimental order parameters were analyzed using a mean-torque potential model for the acyl chain segment distributions, and comparison was made with the conventional diamond lattice approach. Statistical mechanical principles were used to relate the measured order parameters to the lipid bilayer structural parameters: the hydrocarbon thickness and the mean interfacial area per lipid. At fixed temperature, the area decreases with increasing acyl length, indicating increased van der Waals attraction for longer lipid chains. However, the main effect of increasing the acyl chain length is on the hydrocarbon thickness rather than on the area per lipid. Expansion coefficients of the structural parameters are reported and interpreted using an empirical free energy function that describes the force balance in fluid bilayers. At the same absolute temperature, the phosphatidylcholine (PC) series exhibits a universal chain packing profile that differs from that of phosphatidylethanolamines (PE). Hence, the lateral packing of phospholipids is more sensitive to the headgroup methylation than to the acyl chain length. A fit to the area per lipid for the PC series using the empirical free energy function shows that the PE area represents a limiting value for the packing of fluid acyl chains.     

Photoinhibition in differently coloured juvenile leaves of Syzygium species

Photoinhibition, as measured by the dark-adapted chlorophyll a fluorescence ratio Fv/Fm, was assessed in Syzygium moorei, a species with dark green juvenile leaves, Syzygium corynanthum, which has light green juvenile leaves, and two species with pink-red juvenile leaves (Syzygium wilsonii and Syzygium luehmannii). All plants were glasshouse-grown (maximum PPFD 1500 mol m^-2 s^-1) under optimum nutrition and water.When measured at midday, dark-adapted Fv/Fm ratios of juvenile leaves gradually increased in all species as percentage of full leaf expansion (% FLE) increased. Fluorescence measurement 3h after sunset or pre-dawn also showed a developmental effect on Fv/Fm, with juvenile leaves of S. luehmannii and S. wilsonii showing much lower Fv/Fm at all stages of development. Dark-adapted Fv/Fm, values in both juvenile and mature leaves generally never exceeded 0.8 at any stage in any of the species.Courses of Fv/Fm on sunny days showed greater diurnal photoinhibition in green juvenile (c. 50% FLE) leaves of S. moorei (24%) and S. corynanthum (36%) than in mature leaves of the previous flush in these species (<10%). Diurnal photoinhibition was statistically similar (18-24%) in pink-red juvenile and green mature leaves of S. luehmannii and S. wilsonii. Re-positioning juvenile leaves of S. wilsonii horizontally increased diurnal photoinhibition.Exposure of leaves to a standard mild photoinhibitory light treatment (30 min at 1000 mol m^-2s^-1) showed that juvenile leaves of all species had a lower percentage of high energy state quenching (qE) and a higher percentage of photoinhibitory quenching (qI) than mature leaves.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

A comparison of the development and metabolic activity of mycorrhizas formed by arbuscular mycorrhizal fungi from different genera on two tropical forage legumes

 Two glasshouse experiments were done to assess the development and metabolic activity of mycorrhizas formed by isolates of arbuscular mycorrhizal fungi (AMF) from three different genera, Acaulospora, Gigaspora and Glomus on either Pueraria phaseoloides L. or Desmodium ovalifolium L. plants. The second of the two experiments included three levels of a localised phosphate source in the pots. Alkaline phosphatase (ALP), stained histochemically in the intra-radical mycelium (IRM) of AMF over sequential harvests, did not provide a direct marker for the efficiency of AMF in mobilising phosphorus (P) for plant growth and development. The ability of the extra-radical mycelium (ERM) to scavenge a localised phosphate source, determined by its extraction from buried 35-μm mesh pouches, was dependent on the species of AMF tested. This work indicates that AMF from different genera have unique patterns of mycelial development when forming mycorrhizas with tropical hosts in the presence of a localised phosphate source. AMF also appear to have different mechanisms for the control of P transfer, within the mycelium, to the host. The significance of the architecture of the ERM is discussed as well as the localisation of ALP in the IRM in determining the efficiency of AMF in terms of P accumulation in planta and subsequent growth of plants. Accepted: 19 August 1998     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

产超广谱β- 内酰胺酶肺炎克雷伯菌耐药性及其基因分型分析

目的:了解新疆独山子地区肺炎克雷伯菌超广谱β-内酰胺酶(ESBLs)的发生率、作为指示剂的五种抗生素的检出情况及ESBLs主要基因型。方法:收集临床分离的148株肺炎克雷伯菌,采用双纸片协同筛选法、NCCLS推荐的表型筛选和确证试验对细菌进行ESBLs产酶株的识别;耐药基因的质粒重组、转化,聚合酶链反应(PCR)扩增阳性产物测序,通过GenBank对序确定基因型。结果:本地区肺炎克雷伯菌ESBLs的分离率达31.1%,头孢曲松(CRO)安曲南(ATM)和头孢噻肟(CTX)头孢泊肟(CPD)、头孢他定(CAZ)作为指示剂检出率分别为97.8%、95.6%、93.4%、76.0%、65.2%;本地区产ESBLs菌株耐药基因型CTX-M-22占54.3%,CTX-M-18占41.3%,TEM61.8%,52.1%的产ESBLs肺炎克雷伯菌SHV耐药基因阳性。结论:CRO、ATM和CTX对检测ESBLs阳性率较高;CTX-M-22、CTX-M-18是本地区产ESBLs菌株的主要基因型。     

小鼠淋巴细胞抗原CTLA-4的胞外肽段表达及纯化

目的: 真核细胞表达小鼠淋巴细胞抗原CTLA-4胞外段肽,研究表达肽段与抗原呈递细胞B7分子结合后减轻小鼠淋巴细胞刺激后的增殖抑制,从而启动T淋巴细胞进一步增殖。方法:从小鼠脾脏淋巴细胞获得总RNA,通过逆转录PCR扩增出CTLA-4全长基因,克隆并测序。依据胞外段序列和真核表达载体pcDNA3.1序列,合成引物扩增胞外片段,两者经内切核酸酶处理、连接构建重组表达pcDNA3.1载体,重组质粒经测序验证后,采用lipofectamine 2000转染入小鼠肝癌细胞Hepa1-6,经G418筛选获得稳定表达细胞株。结果:获得小鼠CTLA-4胞外段真核表达载体和小鼠肝癌细胞Hepa1-6稳定表达转染细胞株,制备了CTLA-4胞外肽段,经His标签抗体和小鼠CTLA-4抗体Western blot检测表达蛋白带均呈阳性。结论:获得CTLA-4胞外段肽,为进一步研究该肽的作用打下基础。