Selective fluorophore-assisted light inactivation of voltage-gated calcium channels

Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca_v1.2 (L-type) and Ca_v3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca_v1.2 calcium channels containing EYFP-labeled α_1C subunits but does not affect the EYFP-α_1G Ca_v3.1 calcium channels or Ca_v1.2 channels containing EYFP-labeled β subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.     

新疆甘草属的种间杂交

试验以居群为单位的新疆甘草属７个种进行种间人工杂交。通过对种间杂交结实量（ＲＳ）和亲本种平均结实量（ＲＳｍ和ＲＳｆ）的分析比较,测出相应的杂交结实指数（ＩＳ）、初步了解种间杂交亲和性的大小,为甘草属植物系统与演化的研究提供参考依据。     

人细胞差异表达基因cDNA消减杂交体系的建立

为克隆肺腺癌分化相关基因, 采用诱导分化与消减杂交相结合的策略, 建立了全反式维甲酸(RA)诱导前后人肺腺癌细胞系的cDNA消减文库, 得到124个cDNA消减克隆. 经加减法杂交差异筛选、DNA和RNA印迹、cDNA全序列测定和生物学功能分析, 分离到3个在人肺腺癌细胞系分化过程中由RA激活而特异表达的新的cDNA序列这一策略和技术路线适用于分离细胞中呈过量表达或表达抑制基因的cDNA克隆, 并具有反映细胞分化过程中基因表达动态变化特征和相对简便适用的特点.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Dynamic properties of the advertisement calls of gray tree frogs: patterns of variability and female choice

We assessed the potential for several acoustic properties ofthe advertisement calls of male gray tree frogs to affect relativemating success by relating patterns of variation in these propertiesto minimum differences required to elicit female choice. Dynamicproperties (pulse number, PN; call rate, CR; and duty cycle,DC, the ratio of call duration to call period) varied much morewithin bouts of calling than a static property (dominant frequency,DF) but nevertheless exhibited significant between male variationin three of four breeding seasons. Many multiply recorded malesconsistently produced calls with values substantially aboveor below mean values of males recorded on the same nights. Nightlyranges of variation in PN and CR were often greater than theminimum differences required to elicit female preferences inthe laboratory. In most experiments, females chose high-PN orfast-CR calls over low-PN or slow-CR alternatives, respectively,even if the preferred stimuli were farther away or 6-10 dB lowerin sound pressure level (SPL), provided that differences inPN or CR were 100%. Consistent with these results, females didnot always choose the closer of two calling males in the field.Nightly ranges of variation in DF rarely equaled the minimumdifference required to elicit SPL independent preferences. Femalespreferred a stimulus of high-PN and slow-CR over an alternativeof low-PN or fast-CR with the same acoustic on-time; in twoexperiments, females chose calls of high-PN over low-PN alternativeseven though the playback of the high-PN call was interruptedand the low-PN call was broadcast continuously. Thus, femalepreferences were not merely based on the total time of acousticstimulation. Responses of females tested twice in the same experimentsuggest that phenotypic variation in preference was limitedin our study populations.     

枳壳外植体离体再生及农杆菌介导的遗传转化

以枳壳实生苗的上胚轴及茎段为材料,在附加有ＢＡ和ＭＴ培养基上进行培养,上胚轴出芽率普遍高于茎段,ＢＡ为１ｍｇ／Ｌ时,出芽率最高,ＢＡ浓度升高,出芽率随之下降。外植体在培养基上的接种方式,对出芽有一定影响,上胚轴切段 形态学下端垂直插入,出芽率高。     

Genetic markers distinguishing between the two subspecies of the rosy bitterling,Rhodeus ocellatus (Cyprinidae)

Eleven populations of the rosy bitterling,Rhodeus ocellatus, from different localities in Japan, were genetically compared at 16 protein-coding loci using starch-gel electrophoresis. Two loci,Ldh-2 andPgdh, were demonstrated as diagnostic markers for the identification of two subspecies;R. ocellatus kurumeus, which is native to Japan, andR. ocellatus ocellatus, which was introduced from China. The two subspecies were distinguished by the complete substitution of different alleles between them. Population ofR. ocellatus kurumeus occurring in Yao City, Osaka, and in Kanzaki, Saga Prefecture were genetically closely related to each other (genetic distance: D=0.056) but distantly so toR. ocellatus ocellatus from Saitama Prefecture (D=0.202 or 0.265). Electrophoretic analyses also elucidated the existence of hybrid populations of the two subspecies. The populations ofR. ocellatus kurumeus in Yao City had lower genetic variability and a lower incidence of white coloration on the ventral fins than populations of the same in Saga Prefecture. The present study strongly implies that the introduction of the foreign freshwater fishes with subspecific differentiation, into the original range of indigenous subspecies, should be averted not to bring the genetic pollution.     

The mitochondrial inner membrane anion channel is inhibited by DIDS

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg²⁺ and H⁺. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC₅₀ values of 26, 44, and 88 M for DIDS, H₂-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl^– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC₅₀ which is severalfold higher than that for malonate. The IC₅₀ for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC₅₀ by about 50%, but under conditions where only the matrix pH is made alkaline the IC₅₀ is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl^– channels than previously thought, but also suggest that other Cl^– channels may be sensitive to some of the many regulators of IMAC which have been identified.     

Conformations Consistent with Charge Migration Observed in DNA and RNA X-ray Structures

Abstract

Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning.     

Three new hybrids of Ophioglossum (Ophioglossaceae) from Monte Pisano,Tuscany (Central Italy)

Based on general morphology, spore measurements and ornamentation (scanning electron microscope), genome size estimation, and molecular systematics (trnL-trnF IGS), we show the extreme systematic complexity within the European representatives of the genus Ophioglossum. In particular, three hybrids from Tuscany are described: the tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane, seen as the hybrid between diploid O. lusitanicum L. and hexaploid O. azoricum C.Presl; the tetraploid O. × giovanninii Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the homoploid hybrid between tetraploid O. vulgatum L. and tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane; the pentaploid O. × pseudoazoricum Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the hybrid between hexaploid O. azoricum C.Presl and tetraploid O. vulgatum L. All the three new taxa grow in different localities in the Monte Pisano mountain range.