Site of action of a gonococcal growth inhibitor produced by Staphylococcus haemolyticus

The inhibitory substance produced by Staphylococcus haemolyticus strain no. 7 acts on growing as well as resting gonococcal cells, as shown by reductions in viable counts. The optical density of these cell suspensions was only slightly reduced. The inhibitor caused lysis of gonococcal spheroplasts at 24 degrees C and 37 degrees C, but was much less active at 4 degrees C. Acting on intact gonococcal cells, the inhibitor caused a temperature-dependent release of radioactive cytoplasmic material. Electronmicroscopy showed that treated suspensions contained ghost cells with the cell envelope relatively intact. Our results suggest that the inhibitor may act on the cytoplasmic membrane of the gonococcal cell causing cytoplasmic leakage and, eventually, death.     

α-干扰素加软坚散结汤抗慢性乙型肝炎肝纤维化及早期肝硬化的临床研究

目的:探讨慢性乙型肝炎肝纤维化及早期肝硬化的中西医结合疗法。方法:采用α-干扰素加中药软坚散结汤联合治疗经病理确诊的慢性乙型肝炎患者62例,并以同期64例慢性乙肝患者作对照,疗程均为6个月。结果:治疗组临床症状及体征明显改善,血清ALT、TBil、A/G、γ球蛋白(γ-GT)和肝纤维化指标水平明显降低,血清白蛋白(A)水平有不同程度升高(P<0.01或0.05);B超示肝内光点粗、Ⅲ级血管走行欠清和分布不均等异常声像图明显改善,与对照组比较差异有显著性意义(P<0.05)。此外,治疗组HBeAg转阴率为61.0％,明显优于对照组36.3％(P<0.01)。结论:α-扰素和中药软坚散结汤联合应用,对改善慢性乙型肝炎患者临床症状体征,促进肝功能复常,抗肝纤维增生和抑制乙肝病复制等有显著疗效。     

Monosodium glutamate-induced reductions in hypothalamic beta-endorphin content result in mu-opioid receptor upregulation in the medial preoptic area.

Estradiol valerate (EV) treatment in the rat induces a lesion of the hypothalamic arcuate nucleus, resulting in significant decreases in hypothalamic beta-endorphin. In addition, the EV treatment causes a selective increase in mu-opioid binding in the medial preoptic area (MPOA). Since beta-endorphin neurons located in the arcuate nucleus project extensively to the MPOA, we have hypothesized that the EV-induced loss of these afferents induces a compensatory upregulation of mu-opioid receptors in opioid target neurons. In order to test this hypothesis, we have utilized monosodium glutamate (MSG) treated animals as a model of beta-endorphin cell loss and hence of beta-endorphin deafferentation of the MPOA. Neonatal MSG treatment has been shown to result in the destruction of 80-90% of arcuate neurons accompanied by pronounced decreases in beta-endorphin concentrations in both arcuate nucleus and MPOA. mu-Opioid binding sites were radioautographically labeled in sections from the MPOA of sham- and MSG-injected animals using the methionine enkephalin analogue 125I-FK 33-824 and quantitated by computer-assisted densitometry. The remainder of the hypothalamus of these same animals was utilized for the determination of the beta-endorphin concentration. The hypothalami of rats treated with MSG exhibited 62% (p < 0.01) less beta-endorphin than saline-injected controls. In addition, the mean mu-opioid-binding densities in the MPOA were 24% (p < 0.05) above controls in the MSG-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)     

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G + C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci. © 1994 Wiley-Liss, Inc.     

Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics

Somatic chromosomal mosaicism is a well-established cause for birth defects, mental retardation, and, in some instances, specific genetic syndromes. We have developed a clinically validated, targeted BAC clone array as a platform for comparative genomic hybridization (aCGH) to enable detection of a wide range of pathologic copy number changes in DNA. It is designed to provide high sensitivity to detect well-characterized submicroscopic micro-deletion and duplication disorders while at the same time minimizing detection of variation of uncertain clinical significance. In the course of studying 2,585 samples submitted to our clinical laboratory, chromosomal mosaicism was detected in 12 patient samples; 10 of these cases were reported to have had a normal blood chromosome analysis. This enhanced ability of aCGH to detect mosaicism missed by routine chromosome analysis may be due to some combination of testing multiple cell lineages and/or failure of cytogenetically abnormal T lymphocytes to respond to mitogens. This suggests that aCGH may detect somatic chromosomal mosaicism that would be missed by conventional cytogenetics.     

Apolipoprotein C-III-1 activates lysosomal sphingomyelinase in vitro.

Apolipoprotein (apo)C-III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 microM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 microM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine three- to fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of 5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.     

Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1.

Gene targeting was used to produce mice deficient in intercellular adhesion molecule 1 (ICAM-1) or CD54, an immunoglobulin-like cell adhesion molecule that binds beta 2 integrins. Homozygous deficient animals develop normally, are fertile, and have a moderate granulocytosis. The nature of the mutation, RNA analysis, and immunostaining are consistent with complete loss of surface expression of ICAM-1. Deficient mice exhibit prominent abnormalities of inflammatory responses including impaired neutrophil emigration in response to chemical peritonitis and decreased contact hypersensitivity to 2,4-dinitrofluorobenzene. Mutant cells provided negligible stimulation in the mixed lymphocyte reaction, although they proliferated normally as responder cells. These mutant animals will be extremely valuable for examining the role of ICAM-1 and its counterreceptors in inflammatory disease processes and atherosclerosis.     

Dermal and pulmonary inflammatory disease in E-selectin and P-selectin double-null mice is reduced in triple-selectin-null mice

In the initial phase of an inflammatory response, leukocytes marginate and roll along the endothelial surface as a result of adhesive interactions between molecules on the endothelial cells and leukocytes. To evaluate the role of the 3 selectins (E, L, and P) in leukocyte rolling and emigration, a null mutation for L-selectin was introduced into previously described embryonic stem cells with null mutations in the genes for both E-selectin and P-selectin (E/P double mutants) to produce triple-selectin-null mice (E-selectin, L-selectin, and P-selectin [E/L/P] triple mutants). Triple-selectin homozygous mutant mice are viable and fertile and only rarely develop the severe mucocutaneous infections or pulmonary inflammation characteristic of E/P double-mutant mice. Surface expression of L-selectin was undetectable in triple-mutant mice on fluorescence-activated cell-sorter analysis of peripheral neutrophils. Pathological studies revealed moderate cervical lymphadenopathy and lymphoplasmacytic infiltrate, but these were less extensive than in E/P double-mutant mice. Neutrophil emigration during thioglycolate-induced peritonitis was significantly reduced at 4, 8, and 24 hours (35%, 65%, and 46% of wild-type values, respectively). Intravital microscopy of the cremaster muscle revealed almost no rolling at times up to 6 hours after exteriorization, with or without addition of tumor necrosis factor alpha. The small amount of residual rolling was dependent on alpha(4)-integrin. The occurrence of skin and pulmonary disease in E/P double-mutant mice but not E/L/P triple-mutant mice suggests that deficiency of L-selectin alters the inflammatory response in E/P mutants. (Blood. 2001;98:727-735)     

A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.

Previously, a hypomorphic mutation in CD18 was generated by gene targeting, with homozygous mice displaying increased circulating neutrophil counts, defects in the response to chemically induced peritonitis, and delays in transplantation rejection. When this mutation was backcrossed onto the PL/J inbred strain, virtually all homozygous mice developed a chronic inflammatory skin disease with a mean age of onset of 11 weeks after birth. The disease was characterized by erythema, hair loss, and the development of scales and crusts. The histopathology revealed hyperplasia of the epidermis, subcorneal microabscesses, orthohyperkeratosis, parakeratosis, and lymphocyte exocytosis, which are features in common with human psoriasis and other hyperproliferative inflammatory skin disorders. Repetitive cultures failed to demonstrate bacterial or fungal organisms potentially involved in the pathogenesis of this disease, and the dermatitis resolved rapidly after subcutaneous administration of dexamethasone. Homozygous mutant mice on a (PL/J x C57BL/6J)F1 background did not develop the disease and backcross experiments suggest that a small number of genes (perhaps as few as one), in addition to CD18, determine susceptibility to the disorder. This phenotype provides a model for inflammatory skin disorders, may have general relevance to polygenic human inflammatory diseases, and should help to identify genes that interact with the beta2 integrins in inflammatory processes.