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61.
Christon J. Hurst Janet C. Blannon Rachel L. Hardaway Wesley C. Jackson 《Applied microbiology》1994,60(9):3462-3465
This study examined whether the practice of incorporating either tetrazolium red or tetrazolium violet dye into plaque assay medium deleteriously influences plaque assay titers. Representative members of six different virus families were studied: Cystoviridae (ϕ6), Leviviridae (MS2), Microviridae (ϕX174), Myoviridae (T2), Podoviridae (P22), and Siphoviridae (Denver, T1, and VD13). Each of the members of the Podoviridae and Siphoviridae families appeared to be suppressed by either one or both dyes at a 300-μg/ml concentration. The chosen representatives of the other bacteriophage families were not suppressed by either dye at a 300-μg/ml concentration. Subsequent trials revealed no suppression of Podoviridae or Siphoviridae plaque assay titers when members of these virus families were tested with the same two dyes at the lower concentrations of 150 and 50 μg/ml. Interestingly, the bacteriophage families whose members were affected by the dyes have additional commonality in that they are the two bacteriophage families whose members possess both double-stranded DNA genomes and noncontractile tails. 相似文献
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63.
Ellen C. Breen Ronald A. Ignotz Laura McCabe Janet L. Stein Gary S. Stein Jane B. Lian 《Journal of cellular physiology》1994,160(2):323-335
This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc. 相似文献
64.
Marc Yudkoff Yevgeny Daikhin Zhi-Ping Lin Liana Nissim Janet Stern David Pleasure Itzhak Nissim 《Journal of neurochemistry》1994,62(3):1192-1202
Abstract: The aim was to study the extent to which leu-cine furnishes α-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 16N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [16N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ~50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ~17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate. 相似文献
65.
Stress-Induced Sensitization of Dopamine and Norepinephrine Efflux in Medial Prefrontal Cortex of the Rat 总被引:8,自引:1,他引:7
Paul J. Gresch †Alan F. Sved †Michael J. Zigmond Janet M. Finlay 《Journal of neurochemistry》1994,63(2):575-583
Abstract: We examined whether prior exposure to chronic cold (17–28 days, 5°C) alters basal or stress-evoked (30-min tail shock) catecholamine release in medial prefrontal cortex, nucleus accumbens, and striatum, using in vivo microdialysis. Basal norepinephrine (NE) concentrations in medial prefrontal cortex did not differ between chronically cold-exposed rats and naive control rats (2.7 ± 0.3 vs. 2.5 ± 0.2 pg/20 µl, respectively). Basal dopamine (DA) efflux in any of the brain regions was not significantly different between chronically cold-exposed rats and naive rats. However, a trend for lower basal DA efflux in the cold-exposed relative to naive rats was observed in medial prefrontal cortex (1.5 ± 0.2 vs. 2.2 ± 0.3 pg/20 µl, respectively), nucleus accumbens (3.7 ± 0.8 vs. 5.4 ± 0.9 pg/20 µl, respectively), and striatum (4.4 ± 0.5 vs. 7.2 ± 1.5 pg/20 µl, respectively). In medial prefrontal cortex of rats previously exposed to cold, tail shock elicited a greater increase from baseline in both DA and NE efflux relative to that measured in naive rats (DA, 2.3 ± 0.3 vs. 1.2 ± 0.1 pg, respectively; NE, 3.8 ± 0.4 vs. 1.4 ± 0.2 pg, respectively). However, in nucleus accumbens or striatum of rats previously exposed to cold, the stress-induced increase in DA efflux was not significantly different from that of naive rats (nucleus accumbens, 1.8 ± 0.7 vs. 1.5 ± 0.3 pg, respectively; striatum, 1.9 ± 0.4 vs. 2.6 ± 0.7 pg, respectively). Thus, both cortical NE projections and cortically projecting DA neurons sensitize after chronic exposure to cold. In contrast, subcortical DA projections do not sensitize under these conditions. 相似文献
66.
The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non-O1 serotypes. 相似文献
67.
The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization 总被引:2,自引:0,他引:2
Si Lok Joseph L. Kuijper Laura J. Jelinek Janet M. Kramer Theodore E. Whitmore Cindy A. Sprecher Shannon Mathewes Francis J. Grant Shaula H. Biggs Gary B. Rosenberg Paul O. Sheppard Patrick J. O''Hara Donald C. Foster Wayne Kindsvogel 《Gene》1994,140(2):203-209
Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns. 相似文献
68.
69.
Enzymeimmunoassays (EIAs) for estrone conjugates (EC), pregnanediol-3-glucuronide (PDG), and C-19 and C-21 progesterone metabolites (C-19/C-21) were used to analyze urine samples from four nondomestic equid species, four tapir species, and two rhinoceros species in an attempt to identify if these assays could be used for diagnosing and monitoring pregnancy. The same urine samples were also analyzed for the presence of equine chorionic gonadotropin (eCG) activity, using a field dipstick test and a radioimmunoassay (RIA). The EC EIA was validated for three equid species and the Malayan tapir. Neither the PDG nor the C-19/C-21 EIAs were validated in any species evaluated. In equid species, the EC EIA demonstrated a specificity (the percentage of nonpregnant samples identified correctly) of 100% and a sensitivity (the percentage of pregnant samples identified correctly) of ≥ 88%. With the exception of the Grevy's zebra, the C-19/C-21 EIA showed a similar accuracy in identifying pregnant and nonpregnant equids. The PDG EIA was not sufficiently accurate to merit its use in equids or tapirs for pregnancy diagnosis. From the data collected, it appears analysis of a single urine by both the EC EIA and the C-19/C-21 EIA would be the best method of pregnancy detection during the last 2 trimesters of gestation, in equid species. In tapirs, the C-19/C-21 EIA was slightly more accurate for pregnancy diagnosis than the EC EIA. The C-19/C-21 EIA had a specificity of 93%, but a sensitivity of only 73% in tapir species. None of the EIAs evaluated demonstrated a sufficient specificity or sensitivity to be useful, as presently performed, for pregnancy diagnosis from a single sample in the black rhinoceros. The eCG dipstick used in this study did not prove a sufficiently reliable test for routine pregnancy in nondomestic equids. The eCG RIA results in the Przewalski's horses and the Hartman's mountain zebra were positive early in gestation, and indicate that gonadotropin analysis may be useful for pregnancy detection in these species. Only very low amounts of eCG activity was measured by the eCG RIA in the tapir and rhinoceros urine samples. © 1994 Wiley-Liss, Inc. 相似文献
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