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991.
Microtiter format gene quantification by covalent capture of competitive PCR products: application to HIV-1 detection. 总被引:2,自引:0,他引:2
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We have developed a simple gene quantification system using the competitive polymerase chain reaction (CPCR) followed by microtiter format analysis. CPCR is carried out using a mutant competitor with the same size as the target DNA product, and a minimal base exchange to insure the same amplification kinetics. One primer is aminated at the 5' end to produce PCR products that are captured onto carboxylated wells of microtiter plates through peptide bond formation. The non-aminated DNA strands are stripped off from the wells by alkali washing, and the remaining aminated strands are hybridized with either a digoxigenin-labeled wild type-specific oligonucleotide probe or a competitor-specific probe. To standardize the hybridization conditions of the probes, a DNA construct containing wild type and mutant competitor sequences in tandem is captured at different concentrations, hybridized with the probes, and used to generate a standard curve. Bound probes are detected by anti-digoxigenin antibody conjugated with peroxidase and chromogen. Optical densities are recorded with a conventional microtiter plate reader and converted to concentrations according to the standard curves. The ratios of wild type DNA to mutant competitor are used to determine the initial amounts of wild type DNA in the samples. This method was used successfully to quantify human immunodeficiency virus type 1 (HIV-1) env gene in human lymphocytes. It only requires a thermal cycler and a conventional microtiter plate reader, and can be readily done on a large scale. Potential applications include detection of other pathogens, diagnosis of genetic disorders and studies of gene expression. 相似文献
992.
993.
Akihide Nakao Tsuyoshi Watanabe Shigeo Taniguchi Motonao Nakamura Zen-Ichiro Honda Takao Shimizu Kiyoshi Kurokawa 《Journal of cellular physiology》1993,155(2):257-264
Prostaglandin (PG) F2α increased [3H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with ED50 values of 2.0 × 10?8 M, 4.6 × 10?8 M, and 7.5 × 10?8 M, respectively. The increase in [3H]thymidine incorporation with PGF2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca2+]i increase with PGF2α was still obvious in the absence of extracellular Ca2+ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF2α, which was sensitive to GTPγS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF2α-specific Cl? current when examined by voltage clamp. This Cl? current was also insensitive to IAP pretreatment and not affected by extracellular Ca2+ concentration ([Ca2+]o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca2+ via an IAP-insensitive G-proteins(s), 2) that this PGF2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc. 相似文献
994.
Hirokazu Tsukaya Taku Takahashi Satoshi Naito Yoshihumi Komeda 《Molecular & general genetics : MGG》1993,237(1-2):26-32
Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes. 相似文献
995.
Taichi Usui Masahiro Suzuki Toshinari Sato Hirokazu Kawagishi Kyoko Adachi Hiroshi Sano 《Glycoconjugate journal》1994,11(2):105-110
Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man
d-mannose
- (M
n) (n=1–5)
-linkedn-mer of mannose
- GlcNAc2
2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose 相似文献
996.
Laboratory experiments were conducted to examine whether the lizardEumeces okadae can visually discriminate between ground beetles with different chemical defenses and color patterns.Harpalus spp., which are uniformly black and emit formic acid, andDischissus mirandus, which has four bright yellow spots on a jet black back and emits metacresol, were tested. TheHarpalus beetles were attacked immediately after presentation, whereasD. mirandus was ignored on sight. By artificially paintingD. mirandus all black, a significantly higher frequency of tongue licks (close chemical examination of prey) was clicited than that by
normally colored control beetles, indicating that the conspicuous coloration ofD. mirandus was functionally and visually warning to the lizards. 相似文献
997.
Hirotoshi Ifuku Kohachi Taniguchi Hisahiro Matsumoto 《European journal of applied physiology and occupational physiology》1994,69(3):244-249
In earlier studies we have shown that both the pressure (P) of the carotid artery pulse (CAP) and its first derivative (CAP dP/dt) could be recorded during moderate exercise. To establish that the CAP (dP/dt)/P is a noninvasive substitute for the left ventricular (LV) value, LV (dP/dt)/P, an index of cardiac contractility, we studied CAP (dP/dt)/P under various states of activity in the autonomic nervous system in 12 healthy male subjects. Increased sympathetic nerve activities yielded by passive tilting, emotional load, or cold stress increased CAP (dP/dt)/P significantly (P< 0.05). Increased parasympathetic nerve activity by ocular compression, however, did not significantly affect the value. Moderate exercise at a heart rate of approximately 150 beats·min–1 increased it significantly from 16.7 to 25.2·s–1 in a supine position (P<0.001) and from 16.6 to 24.8·s–1 in an upright position (P<0.001). It increased monotonically as heart rate increased, but the slope was steeper when the heart rate was greater than approximately 100 beats·min–1 than it was when the rate was less than 100 beats·min–1. In conclusion, the present study indicated that CAP (dP/dt)/P can be used as a noninvasive index of cardiac contractility even in moderate exercise. 相似文献
998.
Z. Radák K. Lee W. Choi S. Sunoo T. Kizaki S. Oh-ishi K. Suzuki N. Taniguchi H. Ohno K. Asano 《European journal of applied physiology and occupational physiology》1994,69(5):392-395
The effects were examined of 6-month intermittent hypobaric (4000 m) exposure on the antioxidant enzyme systems in soleus and tibialis muscles of rats. At the end of the 6-month experimental exposure, the six rats in both the exposed group and the control group were sacrificed. Immunoreactive mitochondrial superoxide dismutase (Mn-SOD) contents were measured as well as the activities of antioxidant enzymes [Mn-SOD, cytosolic SOD (Cu,Zn-SOD), catalase (CAT), and glutathione peroxidase (GPX)]. Thiobarbituric acid-reactive substances (TBARS) were also determined as an indicator of lipid peroxidation. The high altitude exposure resulted in a marked increase in TBARS content in soleus muscle, suggesting increased levels of oxygen free radicals. Conversely, significant decreases in both Mn-SOD content and activity in solens muscle were oted affer exposure. Such trends were not noticed in tibialis muscle. On the other hand, no significant changes in Cu,Zn-SOD, CAT, or GPX were observed in either muscle. These results suggested that the increases in lipid peroxidation were most probably a result of decreased Mn-SOD function which was more depressed in oxidative than in glycolytic muscle. 相似文献
999.
Atsuhiko Ishida Hirokazu Kanefusa Hidenori Fujita Tetsuo Toraya 《Archives of microbiology》1994,161(4):293-299
Novel vitamin B12 analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B12- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5.6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B12 activity and behaved as weak B12 antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. ON the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [3H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B12 activity toward E. coli 215 by blocking the B12-transport systemAbbreviations AdoCbl
adenosylcobalamin
- MeCbl
methylcobalamin
- CN-Cbl
cyanocobalamin or vitamin B12
- Cbl
cobalamin
- (CN, aq)Cbi
cyanoaquacobinamide
- MeCbi
methylcobinamide
- Cbi
cobinamide
- (CN, aq)Cbi-PMe
cyanoaquacobinamide methyl phosphate
- Cbi-PMe
cobinamide methyl phosphate
- DBI
5,6-dimethylbenzimidazole
- DBIyl
5,6-dimethylbenzimidazolyl
- FMNH2
fully reduced form of riboflavin 5-phosphate 相似文献
1000.
An enzyme-linked immunosorbent assay (ELISA) system for hepatitis B e antigen (HBeAg) was developed employing beta-D-galactosidase conjugated with antibody to HBeAg (anti-HBe) and using m-maleimidobenzoyl-N-hydroxysuccinimide ester as the coupling reagent. The experimental conditions for quantitative assay of HBeAg were determined. The presence of rheumatoid factor in test sera did not affect the results. This assay system is more sensitive than the micro-Ouchterlony method and as sensitive as radioimmunoassay. The use of beta-D-galactosidase for ELISA in the field of virology is recommended. 相似文献