Role for recombinant gamma-glutamyltransferase from Treponema denticola in glutathione metabolism

Volatile sulfur compounds, including hydrogen sulfide (H(2)S), have been implicated in the development of periodontal disease. Glutathione is an important thiol source for H(2)S production in periodontal pockets. Our recent studies have delineated a pathway of glutathione metabolism in Treponema denticola that releases H(2)S. In this pathway, gamma-glutamyltransferase (GGT) has been proposed to catalyze the first step of glutathione degradation. We have cloned the gene of GGT from T. denticola, which contains an open reading frame of 726 bp encoding a protein of 241 amino acids. Transformation of this gene into Escherichia coli led to the expression of a recombinant protein. After purification by chromatography, the recombinant protein showed enzymatic activity typical of GGT, catalyzing the degradation of Na-gamma-glutamyl-4-nitroaniline (GNA) and the hydrolysis of glutathione, releasing glutamic acid or glutamine and cysteinylglycine. L-Cysteine is not a substrate of GGT. Importantly, GNA, when added to T. denticola, was able to compete with glutathione and inhibit the production of H(2)S, ammonia, and pyruvate. This was accompanied by the suppression of hemoxidative and hemolytic activities of the bacteria. Purified GGT was inactivated by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and proteinase K treatment. However, higher enzymatic activity was demonstrated in the presence of 2-mercaptoethanol and dithiothreitol. Our further experiments showed that the addition of recombinant GGT to Porphyromonas gingivalis, a bacterium without significant glutathione-metabolizing capacity, drastically increased the utilization of glutathione by the bacterium, producing H(2)S, ammonia, and pyruvate. This was again accompanied by enhanced bacterial hemoxidative and hemolytic activities. Together, the results suggest an important role for GGT in glutathione metabolism in oral bacteria.     

大鼠中缝大核突触囊泡在电针后的变化及其图象分析

应用透射电镜和图象分析方法,对电针后大鼠中缝大核突触前终扣内突触囊泡的数量以及突触前终扣内容物的量的变化进行了研究。结果表明:电针后,中缝大核突触前终扣内圆形清亮囊泡和颗粒囊泡的数量明显减少,突触囊泡聚集并向突触活性带集中。图象分析结果:电针后,突触前终扣内容物的量较对照组明显减少,而且内容物分布不均匀并在突触膜附近集中。本实验为针刺镇痛的理论研究提供了形态学依据。     

No direct role for Epstein-Barr virus in American hepatocellular carcinoma

Epstein-Barr virus (EBV) was recently linked to hepatocellular carcinogenesis in Japanese patients. It is not clear whether EBV infection is also associated with hepatocellular carcinoma (HCC) occurring in American patients. We studied 41 cases of HCC from the Los Angeles area for evidence of EBV infection by in situ hybridization, immunohistochemistry, and polymerase chain reaction methods. Of 41 cases, 16 were seropositive for hepatitis B virus surface antigen (39%), 9 of 29 tested were seropositive for hepatitis C virus antibody (31%); in total, 22 cases were seropositive for hepatitis B virus and/or hepatitis C virus (53%). Of 41 cases, 1 was positive for EBV-encoded small nonpolyadenylated RNA (EBER)-1 (2%) by in situ hybridization. By immunohistochemistry, two cases were positive for EBV nuclear antigen (EBNA)-1 (5%), one was positive for the transactivating immediate early BZLF1 (ZEBRA) (2%), and none was positive for latent membrane protein-1. None of the 41 cases was positive for latent membrane protein-1 and EBV nuclear antigen (EBNA)-4 DNAs by polymerase chain reaction assay. All four positive cases showed rare EBER-1-, ZEBRA-, or EBNA-1- positive cells (<0.1%); in none of these cases was there expression of any other EBV viral genes. In the one case each that was positive for EBER-1 and ZEBRA, both of which occurred in patients of non-Asian ethnicity, the staining was limited to infiltrating small lymphocytes, and tumor cells were negative. In the two cases that were positive for EBNA-1, both of which occurred in patients of Asian ethnicity, the staining was limited to tumor cells, and infiltrating small lymphocytes were negative. Our study indicates that rare cases of American HCC may contain EBV-infected cells, but it is unlikely that EBV plays a major role in the carcinogenesis of HCC.     

Carbon plasma immersion ion implantation of nickel-titanium shape memory alloys

Nickel-titanium (NiTi) shape memory alloys possess super-elasticity in addition to the well-known shape memory effect and are potentially suitable for orthopedic implants. However, a critical concern is the release of harmful Ni ions from the implants into the living tissues. We propose to enhance the corrosion resistance and other surface and biological properties of NiTi using carbon plasma immersion ion implantation and deposition (PIII&D). Our corrosion and simulated body fluid tests indicate that either an ion-mixed amorphous carbon coating fabricated by PIII&D or direct carbon PIII can drastically improve the corrosion resistance and block the out-diffusion of Ni from the materials. Our tribological tests show that the treated surfaces are mechanically more superior and cytotoxicity tests reveal that both sets of plasma-treated samples favor adhesion and proliferation of osteoblasts.     

Langerhans' cell expression of the selectin ligand, sialyl Lewis x.

Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues. A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes. As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans. Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9). Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10). E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis. Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x. In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture. CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase. Expression of other selectin ligands was also examined. While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions. These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin.     

人际交往训练降低医学生心理应激反应的实验研究

目的　探讨人际交往训练降低医学生心理应激反应的有效模式 ,以培养交往手段 ,促进身心健康 ,提高学习效率。方法　从云南中医学院中医专业 2 0 0 0级 8个班 ,41 2名学生中随机抽取 2个班 ,共 1 0 6人为被试对象 ,结合专业学习 ,在生理学课堂教学中贯穿人际交往训练 ,训练前后分别测查 90项症状自评量表 ( SCL-90 )和防御方式问卷 ( DSQ) ,并用 Q分类法检验被试课前和课后自我评价。最后 ,对被试和其余 6个班的学生进行生理测验 ,并对测验成绩进行比较。结果　被试在交友信心、乐观豁达、口头表达、积极接纳、说服技巧方面均比训练前有显著和极显著提高 ;且考前心理应激反应明显降低 ,防御方式趋向成熟 ,测验及格率高于其余 6个班。结论　结合专业进行人际交往训练可在近期内改善医学生的心理健康水平 ,有利于提高学习效率 ,深受大学生欢迎     

Extended epidemic of nosocomial urinary tract infections caused by Serratia marcescens

In recent years a significant increase in the incidence of Serratia marcescens infections was noted at the Chang Gung Memorial Hospital, Taoyuan, Taiwan. A review of laboratory (1991 to 2002) and infection control (1995 to 2002) records showed the possibility of an extended epidemic of nosocomial urinary tract infections (UTIs) caused by S. marcescens. Therefore, in 1998 and 1999, 87 isolates were collected from patients with such infections and examined and another 51 isolates were collected in 2001 and 2002. The patients were mostly elderly or the infections were associated with the use of several invasive devices. S. marcescens was usually the only pathogen found in urine cultures in our study. Neither prior infections nor disseminated infections with the organism were observed in these patients. Resistance to most antibiotics except imipenem was noted. Two genotyping methods, pulsed-field gel electrophoresis and infrequent-restriction-site PCR, were used to examine the isolates. A total of 12 genotypes were identified, and 2 predominant genotypes were found in 72 (82.8%) of the 87 isolates derived from all over the hospital. However, 63.9% of the isolates of the two genotypes were from neurology wards. A subsequent intervention by infection control personnel reduced the infection rate greatly. The number and proportion of the two predominant genotypes were significantly reduced among the 51 isolates collected in 2001 and 2002. Thus, a chronic and long-lasting epidemic of nosocomial UTIs caused by S. marcescens was identified and a successful intervention was carried out. Both a cautious review of laboratory and infection control data and an efficient genotyping system are necessary to identify such a cryptic epidemic and further contribute to the quality of patient care.     

Intracellular Abeta is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines.

Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.     

Cell-free translation of tobacco vein mottling virus RNA

Tobacco vein mottling virus (TVMV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system. The RNA was approximately 5% as active as rabbit globin mRNA in directing protein synthesis. Translation was stimulated approximately 15% by the cap analog pppm7G, a phenomenon which has also been observed with uncapped viral RNAs. Treatment with NaIO4 and NaB(3H]4 under conditions which label the caps of globin and ovalbumin mRNA failed to produce labeled cap structures in TVMV RNA. Approximately 20 polypeptides, ranging from 20,000 to 100,000 daltons, were produced in the reticulocyte system programmed with TVMV RNA. The predominant species (P75) had a molecular weight of 75,000. The same major polypeptides were produced in the wheat germ system, but there was relatively greater synthesis of the smaller polypeptides. Incubation of the reticulocyte system in the presence of cycloheximide following an initial period of synthesis failed to cause breakdown of larger polypeptides into smaller polypeptides. Preincubation of TVMV RNA with the reticulocyte system before addition of labeled amino acids caused greater synthesis of the smaller polypeptides, suggesting that they are derived from fragments of the RNA produced during the incubation. Translation of TVMV RNA which had been bound to oligo(dT)-cellulose yielded nearly the same spectrum of polypeptides, but synthesis of polypeptides smaller than 52,000 daltons was reduced. At low ionic strength, only polypeptides of 52,000 daltons and smaller were synthesized. At high ionic strength, P75 was essentially the only product synthesized. Antibody to whole virus precipitated many of the polypeptides, including P75, suggesting that they contain the coat protein sequence. No polypeptide with the electrophoretic mobility of authentic coat protein, however, was precipitated. Comparison of partial proteolytic digests of P75 and authentic coat protein provided further evidence for sequence homology.     

Untersuchungen zum Problem der Mumpsimmunität

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