Ⅱ型限制性核酸内切酶NcrⅠ的研究

 在肉色诺卡氏菌C-212株Nocardia carnea C-212中筛选到一种Ⅱ型限制性核酸内切酶NcrⅠ,经与BglⅡ的λDNA降解物的酶谱比较,以及酶识别特异性和切割位点的检测,证明了NcrⅠ是已知的限制酶BglⅡ的同切限制酶,而且其切割位点也与BglⅡ相同,其为:     

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl₂ at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys¹⁴ to Cys⁷⁹, Cys⁵⁷ to Cys¹⁰⁸, and Cys⁶⁷ to Cys¹¹⁰. This disulfide structure is homologous to the NGF family of neurotrophic factors.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

二核苷酸重复多态性的非同位素检测及其在基因诊断中的应用

本文报道了一种检测二核苷酸重复多态性的简便的非同位素法,利用重复序列两侧的特异引物进行ＰＣＲ扩增,产生的等位片段在薄层变性聚丙烯酰胺凝胶电泳上分离,再用灵敏的银染法显色。该方法不需要标记ＰＣＲ产物,简便、快速,分辨率可达１ｂｐ,并可用多对引物同时进行多重ＰＣＲ分析。用此方法对ＤＭＤ家系成员ｄｙｓｔｒｏｐｈｉｎ基因的５＇－脑型外显子止游区和３＇－非翻译区的两个（ＣＡ）。位点进行了扩增片段长度多态性分     

FIBONACCI级数与菊花结构规律的研究

本文论证了菲波纳斯（Fibonacci）级数与菊花花冠结构的关系,发现了花冠的通用数学模式：花冠（数目）＝（5×倍数）十F_n（F_(n－1)＋F_(n-2)）     

Bi（Ⅲ）与金属硫蛋白作用性质研究

 Ｂｉ（Ⅲ）与金属硫蛋白作用性质研究张保林,黄辉,朱凌燕,岳晟,唐雯霞（南京大学配位化学研究所,配位化学国家重点实验室,南京２１００９３）如何降低顺铂或其它抗癌铂的毒性,一直是癌症化疗中的重要课题之一,最近研究发现预先给大鼠或肺癌病人服用铋盐,可以极大...     

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements.     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

Map locations of mouse hepatitis virus temperature-sensitive mutants: confirmation of variable rates of recombination.

Using standard genetic recombination techniques, studies in our laboratory suggest that recombination rates are very high and vary in different portions of the mouse hepatitis virus (MHV) genome. To determine the actual recombination frequencies in the MHV genome and localize the nucleotide boundaries of individual viral genes, we have sequenced temperature-sensitive and revertant viruses to identify the location of specific mutant alleles. Complementation group F RNA+ ts mutants (LA7, NC6, and NC16) each contained a unique mutation which was tightly linked to the ts phenotype and resulted in a conservative or nonconservative amino acid change in the MHV S glycoprotein gene. In agreement with previous recombination mapping studies, the mutation in LA7 and NC6 mapped within the S1 domain while NC16 mapped within the S2 domain. To determine the map coordinates of the MHV polymerase genes, several RNA- mutants and their revertants belonging to complementation groups C (NC3 and LA9) and E (LA18 and NC4) were also sequenced. Mutations were identified in each virus that were tightly linked to the ts phenotype and resulted in either a conservative or nonconservative amino acid change. The group C allele spanned the ORF 1a/ORF 1b junction, while the group E mutants mapped at the C terminus of ORF 1b about 20 to 22 kb from the 5' end of the genome. Mutation rates, calculated from the reversion frequencies of plaque-purified ts viruses requiring a single nucleotide alteration for reversion, approached 1.32 (+/- 0.89) x 10(-4) substitutions per nucleotide site per round of template copying. Detailed recombination mapping studies across known distances between these different ts alleles has confirmed that homologous recombination rates approached 25% and varied within different portions of the MHV genome.