人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。     

Mechanisms of cardiac cell excitation with premature monophasic and biphasic field stimuli: a model study.

The mechanisms by which extracellular electric field stimuli induce the (re)excitation of cardiac cells in various stages of refractoriness are still not well understood. We modeled the interactions between an isolated cardiac cell and imposed extracellular electric fields to determine the mechanisms by which relatively low-strength uniform monophasic and biphasic field stimuli induce premature reexcitations. An idealized ventricular cell was simulated with 11 subcellular membrane patches, each of which obeyed Luo-Rudy (phase 1) kinetics. Implementing a standard S1-S2 pulse protocol, strength-interval maps of the cellular excitatory responses were generated for rectangular monophasic and symmetric biphasic field stimuli of 2, 5, 10, and 20 ms total duration. In contrast to previously documented current injection studies, our results demonstrate that a cardiac cell exhibits a significantly nonmonotonic excitatory response to premature monophasic and, to a much lesser degree, biphasic field stimuli. Furthermore, for monophasic stimuli at low field strengths, the cell is exquisitely sensitive to the timing of the shock, demonstrating a classic all-or-none depolarizing response. However, at higher field strengths this all-or-none sensitivity reverts to a more gradual transition of excitatory responses with respect to stimulus prematurity. In contrast, biphasic stimuli produce such graded responses at all suprathreshold stimulus strengths. Similar behaviors are demonstrated at all S2 stimulus durations tested. The generation of depolarizing (sodium) currents is triggered by one or more of the sharp field gradient changes produced at the stimulus edges-i.e., make, break, and transphasic (for biphasic stimuli)-with the magnitude of these edge-induced current contributions dependent on both the prematurity and the strength of the applied field. In all cases, however, depolarizing current arises from the partial removal of sodium inactivation from at least part of the cell, because of either the natural process of repolarization or a localized acceleration of this process by the impressed field.     

Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)     

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation.     

不同森林群落结构与光能利用率的关系

本文在人工落叶松纯林及人工落叶松与水典柳混交林的林冠观测数据的基础上建立了落叶松松和水曲柳的树冠锥体模型。通过对上述两种森林群落结构的太阳辐射的观测,利用电磁波的吸收,反射和透射理论分别对以上两种森林群落的光能利用率进行了计算。结果表明理论计算值与实测结果基本一致;双层次混交林的光能利用率高于单层纯林的光能利用率。     

Evidence that 5-HT2 antagonism elicits a 5-HT3-mediated increase in dopamine transmission

Amperozide, a novel atypical antipsychotic drug with few extrapyramidal side effects, is a strong serotonin₂ (5-HT₂) antagonist but has low affinity for dopamine receptors in vitro. The effect of amperozide on the dopaminergic synapse was studied with an in vivo microdialysis technique using anesthetized male Sprague-Dawley rats. Following implantation of dialysis probes into the striatum and nucleus accumbens (NuAc), amperozide was intravenously infused as six consecutive incremental doses (0.5, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/kg) at intervals of 15 min. From the beginning of drug infusion, perfusates were collected in fractions every 30 min throughout a total period of 120 min. The samples were then immediately analyzed by high-performance liquid chromatography with electrochemical detection. Amperozide induced a dose-related elevation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels in both areas.p-Chlorophenylalanine (pCPA) pretreatment abolished the production of 5-HIAA in both areas and attenuated the amperozide-induced rise of DOPAC but not of dopamine. After pretreatment with an intravenous 5-HT₃ antagonist, MDL 72222, the amperozide-induced changes in dopamine, DOPAC and 5-HIAA in both areas were lower than in the saline control group. Preliminary data showed that afterpCPA pretreatment, incremental concentrations of the 5-HT₃ agonist 1-(m-chlorophenyl)-biguanide perfused via the probe also produced significant elevation of dopamine and DOPAC levels in these two areas. Taken together, these results suggest that amperozide may directly block 5-HT₂ receptors in the striatum and NuAc, thereby enhancing 5-HT transmission. The enhanced 5-HT transmission may activate postsynpatic 5-HT₃ receptors located on the dopaminergic terminals, leading to changes in dopamine transmission in these two areas.     

The effect of ethanol on lateral and rotational mobility of plasma membrane vesicles isolated from cultured mar 18.5 hybridoma cells

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to evaluate the effect of ethanol on the rate and range of the lateral mobility and the range of the rotational mobility of bulk bilayer structures of the plasma membrane vesicles (ATCC-PMV) isolated from cultured hybridoma cells (ATCC TIB 216). In a concentration-dependent manner, ethanol increased the excimer to monomer fluorescence intensity ratio (I/I) of Py-3-Py in the ATCC-PMV and decreased the anisotropy (r), limiting anisotropy (r) and order parameter (S) of DPH in the ATCC-PMV. This indicates that ethanol increased both the lateral and rotational mobility of the probes in the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was utilized to examine the range of transbilayer asymmetric rotational diffusion of the ATCC-PMV. The anisotropy (r), limiting anisotropy (r ) and order parameter (S) of DPH in the inner monolayer were 0.024, 0.032, and 0.069, respectively, greater than calculated for the outer monolayer of the ATCC-PMV. Selective quenching of DPH by trinitrophenyl groups was also used to examine the transbilayer asymmetric effects of ethanol on the range of the rotational mobility of the ATCC-PMV. Ethanol had a greater increasing effect on the range of the rotational mobility of the outer monolayer as compared to the inner monolayer of the ATCC-PMV. It has been proven that ethanol exhibits a selective rather than nonselective fluidizing effect within the transbilayer domains of the ATCC-PMV.This paper was supported in part by a research grant from the Korea Science and Engineering Foundation (KOSEF 88-1013-01) and from the Korea Research Foundation (1991–1993).     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.     

Production of panose from maltose by intact cells of Aureobasidium pullulans

Panose, a major component of isomalto-oligosaccharides, was selectively produced from maltose using transglucosylation reaction catalyzed by intact cells of Aureobasidium pullulans. When 50 %(w/v) maltose was used as a substrate, the maximum concentration of panose accumulated in the final reaction mixture was about 50 %(w/w) after 120 hr reaction at 55 °C.