Biochemical and genetic analysis of Streptococcus mutans alpha-galactosidase.

The aga gene coding for alpha-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage lambda. The gene was subcloned into plasmid vectors and shown to specify a novel protein of Mr 80,000. Characterization of alpha-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the alpha-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted Mr of 82,022 and revealed regions of homology to alpha-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all alpha-galactosidase activity and abolished the ability to ferment melibiose; alpha-glucosidase activity was also lost, due to an indirect effect on the dexB gene.     

溶液流动对等电溶菌酶晶体生长的影响

本文对等温自由生长和强制性溶液生长的等电溶菌酶的晶体形态进行了研究,发现这些形态变化与溶液相的流动密切相关,指出生物晶体生长停止是由于生长晶体周围的溶质贫乏造成的;通过某些手段减薄或消除这一溶质贫乏区,就可以保证晶体的持续生长。本文的研究对改善大尺寸晶体的生长提供了一条途径。     

层粘连蛋白与顺铂对腹水肝癌细胞内微丝的共同作用关系

本文探讨在外源性层粘连蛋白与抗癌药物顺铂的共同作用下,癌细胞内微丝组装的变化。结果发现外源性层粘连蛋白与小鼠腹水型肝癌细胞膜受体结合后,促进肌动蛋白微丝组装,使其含量增加;而多靶性抗癌药物顺铂与肌动蛋白微丝的结合,抑制微丝组装过程,造成微丝含量减少;两种试剂共同作用于癌细胞时,肌动蛋白微丝的含量与对照组相比非常接近。本研究为上述两种物质对癌细胞内微丝组装的拮抗性作用提出直接证据。     

HBsAg阴性母亲的新生儿接种不同剂量乙型肝炎血源疫苗的效果

HBsAg阴位母亲的新生儿,按0,1、6月程序分别接种10-10-10μg(1组)、20-10-10μg(2组)和30-10-10μg(3组)乙型肝炎血源疫苗。第一针后一年,检查抗-HBs阳转率,分别为87.60％,90.64％和88.97％,无统计学显著差异。3针10μg组免疫后l～4年抗-HBs阳性率分别为88.31％、81.08％、80.10％和78.39％,虽稍下降,但无统计学显著差异。3个剂量组HBsAg阳性率分别为0.71％,0.49％和0.74％,说明HBsAg阴性母亲的新生儿,用国产血源HBsAg疫苗免疫以10μ×3效果较理想。     

斑点酶免疫实验检测流行性出血热抗体方法的研究

本文应用斑点酶免疫试验(DEIA),检测流行性出血热病人血清42份,其结果与间接免疫荧光(IFA)进行了比较,符合率100％,其DEIA几何干均滴度为1:880,IFA为1:580,同类风湿因子阳性血清[RF(+)]无交叉反应,证明本方法具有较高的敏感性和特异性,本文还对不同方法处理组织内源酶的效果和不同阻断剂的阻断效果进行了比较。试验结果表明本法不但敏感、特异,而且简单、快速、安全、经济、易于推广。     

Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor

We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor''s extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin''s assorted actions on non-platelet cells? We now report that the novel thrombin receptor agonist peptide reproduces thrombin-induced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin- responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA. Our data show that important thrombin-induced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors.     

Differences in the In Vitro and In Vivo 5-Hydroxytryptamine Extraction Performance Among Three Common Microdialysis Membranes

The present study summarizes the results of an in vitro and in vivo comparison of the apparent 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid dialysis performance of three types of membrane frequently used in intracerebral microdialysis experiments. The dialysis fiber types examined were a regenerated cellulose Cuprophan (GF), a proprietary polycarbonate ether (CMA), and a polyacrylonitrile/sodium methallylsulfonate copolymer (HOSPAL). The experiments unexpectedly revealed that the HOSPAL membrane-equipped probes displayed clearly aberrant 5-HT diffusion dynamics compared with GF and CMA probes, demonstrable not only in vitro, but also in in vivo experiments. In vitro, the GF and CMA membrane-equipped probes exhibited maximum relative recovery for 5-HT already in the first 20-min sample, whereas the 5-HT recovery of HOSPAL probes increased in a very slow and protracted manner over a period of a little less than 2 h. The GF and CMA probes further displayed an immediate washout of 5-HT when the probes were subsequently transferred to artificial CSF only-containing medium (no 5-HT), whereas approximately 2 h was required to yield near-total extinction of dialysate 5-HT with the standard HOSPAL probes. In vivo, the rat ventral hippocampal dialysate 5-HT output responses to K+ (100 mM) infusion, to Ca2+ omission, and to systemic 8-hydroxy-2-(di-n-propylamino)tetralin injection were all markedly retarded and blunted when HOSPAL instead of GF membrane-equipped probes were used. However, the 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid extraction in vitro and in vivo were comparable using either of the membrane types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crossover site selection during recombination of polyomavirus replicons.

Two hybrid replicons containing polyomavirus (Py) genomes with large duplications of the viral late coding sequences were transfected into various permissive mouse cell lines. In all cell lines, either replicon yielded the sole amplifiable product expected from intramolecular homologous recombination, unit-length Py DNA (P155). In normal and in Py-transformed cells, such recombination was highly effective and involved sequences previously found to act as recombination hot spots (S repeats). In cells transformed by simian virus 40, however, these hot spots were inoperative in the generation of P155, which occurred with a reduced efficiency. These data confirm and extend earlier data indicating that the nature of products arising from recombination in Py replicons is tightly controlled by both cis- and trans-acting factors.     

Measuring the microelastic properties of biological material.

We have used the atomic force microscope (AFM) to measure the local rigidity modulus at points on the surface of a section of hydrated cow tibia. These data are obtained either from contrast changes that occur as the contact force is altered, or from force versus distance curves obtained at fixed points. These two methods yield the same values for rigidity modulus (at a given point). At low resolution, the elastic morphology and topography mirror the features seen in optical and electron micrographs. At high resolution we see dramatic variations in elastic properties across distances as small as 50 nm.