Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein mRNAs in the peripheral blood leukocytes of asthmatics

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. We have studied the effect of glucose and long chain fatty acids on the HSL mRNA content of 3T3-F442A adipocytes. Exposure of the cells for 32 h to a medium without glucose led to a decrease by a factor of 2.5-3 in the HSL mRNA without a change in C/EBP alpha mRNA or triglyceride content of the adipocytes. The reduction in HSL mRNA level was accompanied by a decrease in HSL total activity. The effect of glucose exposure was observed after 24 h of exposure and was reversible. Treatment of the adipocytes with oleate did not affect HSL expression. These data show that glucose modulates HSL gene expression and hence could participate in the regulation of adipose tissue lipolysis.     

Two isozymes of P450nor of Cylindrocarpon tonkinense: molecular cloning of the cDNAs and genes, expressions in the yeast, and the putative NAD(P)H-binding site

The cDNAs and genes for two isozymes of cytochrome P450nor of the fungus Cylindrocarpon tonkinense, P450nor1 and P450nor2, were cloned and sequenced. Their deduced amino acid sequences respectively showed 83 and 70% identity to that of P450nor of Fusarium oxysporum, and 69% identity to each other. The genes for P450nor1 and P450nor2 were termed, respectively, CYP 55A2 and CYP 55A3. The cDNA for P450nor1 contained a targeting-like presequence upstream the N-terminus of mature protein whereas that for P450nor2 did not, suggesting their different intracellular localisations. We also succeeded in expressing these P450nor isoforms in the host-vector system of the yeast Saccharomyces cerevisiae. We purified one of the recombinant proteins, P450nor of F oxysporum. Little difference could be observed between the native and recombinant proteins in catalytic and spectroscopic properties. We constructed chimeric proteins of P450nor of F oxysporum and P450nor2 which are different in their specificity against the electron donors: reduced pyridine nucleotides. The specificity of chimeric proteins against NADH/NADPH showed that the specificity is determined by the N-terminal half of protein. We found a consensus amino acid sequence between three isoforms of P450nor, A-X-G-X-X-A, similar to the NAD-binding motif G-X-G-X-X-G/A in the region that corresponds to the B'-helix in P450cam.     

Ubiquitin in cerebrospinal fluid: a rapid competitive enzyme-linked immunoflow assay

The aims of this study were to develop a rapid immunoassay to determine the levels of ubiquitin in cerebrospinal fluid and to establish the ubiquitin levels in the spinal fluid of normal aged individuals. A competitive enzyme-linked immunoflow assay was developed. In this assay, ubiquitin is bound to nitrocellulose membrane, after which the primary antibody-test sample mixture and the enzyme-labeled secondary antibody under vacuum are applied sequentially. The final reaction product is collected in a microtiter plate by suction. This competitive assay requires only approximately 4 h and is potentially useful for determining in biological fluids the levels of any antigen or its antibodies that might be present. Employing this immunoassay, cerebrospinal fluid ubiquitin levels were found to be 147.5 +/- 5.2 ng ml-1 in non-neurological aged cases.     

High performance 35 nm gate length CMOS with NO oxynitride gate dielectric and Ni salicide

The 35 nm gate length CMOS devices with oxynitride gate dielectric and Ni salicide have been fabricated to study the feasibility of higher performance operation. Nitrogen concentration in gate oxynitride was optimized to reduce gate current I/sub g/ and to prevent boron penetration in the pFET. The thermal budget in the middle of the line (MOL) process was reduced enough to realize shallower junction depth in the S/D extension regions and to suppress gate poly-Si depletion. Finally, the current drives of 676 /spl mu/A//spl mu/m in nFET and 272 /spl mu/A//spl mu/m in pFET at V/sub dd/=0.85 V (at I/sub off/=100 nA//spl mu/m) were achieved and they are the best values for 35 nm gate length CMOS reported to date.     

Screening the p53 status of human cell lines using a yeast functional assay

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promotor in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30 degrees C but not at 37 degrees C. Four temperature-sensitive p53 mutations were isolated: CAT-->CGT at codon 214 (H214R), TAC-->TGC at codon 234 (Y234C), GTG-->ATG at codon 272 (V272M), and GAG-->AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization.     

Preventive effects of polysaccharides extracted from human tubercle bacilli (specific substance of Maruyama) on colonic carcinogenesis in rats

Polysaccharides extracted from human tubercle bacilli (specific substance of Maruyama [SSM]) have been clinically applied with satisfactory results. Thymidylate synthetase (TS) and thymidine kinase (TK) are key enzymes in de novo and salvage pathways for pyrimidine nucleotide synthesis. Well- and moderately well differentiated adenocarcinomas induced with 1,2-dimethylhydrazine (DMH) are widely distributed throughout the colorectal tract with high TK activity, and the poorly differentiated type is mainly restricted in the proximal colon and the cecum with high TS activity in rats. Subcutaneously injecting the rats with SSM reduced TS activity in colonic nontumorous regions, but in the tumorous regions it reduced TK activity compared with that of the DMH-treated rats without SSM treatment. SSM is suggested to reduce the colorectal carcinogenesis induced with DMH by inhibiting DNA synthesis in a de novo pathway, and to suppress the development of the tumors by decreasing DNA synthesis in the salvage pathway in rats.     

Binding of antibiotics to glycoproteins of the vitelline and fertilization envelopes of cherry salmon eggs

The binding of antibiotics (gentamicin, oleandomycin and chloramphenicol) to vitelline and fertilization envelopes and their extracts was investigated by immunohistochemical and immunocytochemical techniques and immunoblot analysis using mature and artificially activated eggs of the fish Oncorhynchus masou. Binding of antibiotics was detected in the vitelline and fertilization envelope outermost layers, the fertilization envelope inner surface and cortical alveolus exudates, with differences in immunoreactive intensity and deposition. The fertilization envelope outermost layer had the capacity to bind much greater amounts of the antibiotics than the vitelline envelope outermost layer. The greater capacity was caused by the deposition of cortical alveolus exudates, which were known to be responsible for functional roles of protection against bacteria, fungi and noxious materials. Treatment of the vitelline and fertilization envelopes with neuraminidase markedly reduced the binding of gentamicin and chloramphenicol but slightly increased that of oleandomycin; binding of the latter to the vitelline and fertilization envelope outermost layers was considerably reduced after treatment with alpha-fucosidase. Treatment of the two envelopes with alpha-mannosidase, beta-galactosidase or beta-D-glucosaminidase did not cause any alteration in immunoreactive intensity or number of immunoreactive deposits. Immunoblot analysis of the vitelline or fertilization envelope extracts indicated that many of the antibiotic-binding substances were glycoproteins, and several major bands were bound by all three antibiotics. These results suggest that the vitelline or fertilization envelopes may have the ability to protect the egg itself, or the embryo, respectively, by trapping antibiotics, and the trapping may be related to the presence of carbohydrate moieties, such as sialyl or fucosyl residues.     

Plasma concentrations of antipsychotic drugs in psychiatric inpatients

We surveyed a random sample (n = 75) of doctors and dentists at University College Hospital, Ibadan, Nigeria. They were offered anonymous testing for hepatitis B surface antigen (HBsAg), hepatitis Be antigen (HBeAG), antibodies to hepatitis B core antigen (anti-HBc) and to hepatitis C virus (anti-HCV), by enzyme immunoassay. The results suggest a high prevalence of hepatitis B virus (HBV) with a high potential of transmissibility, as well as a high prevalence of HCV infection. The majority of the doctors and dentists use universal precaution for protection against viral hepatitis on < 50% of the occasions when they carry out procedures on their patients. Infection with HBV was associated with type of specialty (surgeons, dentists) and lack of HBV vaccination (p < 0.05). After logistic regression, these factors were independently associated with HBV infection (p < 0.05). Sixty (80%) had not received prior HBV vaccination. Unvaccinated personnel were more likely to be surgeons, dentists, < 37 years of age, and have fewer years of professional activity (p < 0.05). After logistic regression, only fewer years of professional activity remained independently associated with lack of vaccination (p < 0.05). To reduce the occupational exposure of HBV, universal precautions must be rigorously adhered to when the doctors and dentists carry out procedures on their patients, and all health-care workers should be vaccinated with HBV vaccine and the HCV vaccine, when it becomes available.