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101.
The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes. 相似文献
102.
Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases 总被引:6,自引:0,他引:6
R G Taylor M A Lambert E Sexsmith S J Sadler P N Ray D J Mahuran R R McInnes 《The Journal of biological chemistry》1990,265(30):18192-18199
Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation. 相似文献
103.
Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium 总被引:2,自引:0,他引:2
The active uptake system for glutamate in Corynebacterium glutamicum is inducible by growth on glutamate as sole energy and carbon source and is also susceptible to catabolite repression by glucose. The basic level of uptake activity is low in glucose-grown cells (1.5 nmol.mg dry mass-1.min-1), it is intermediate when acetate is the carbon source (3.8 nmol.mg dry mass-1.min-1) and becomes fully induced by glutamate (15 nmol.mg dry mass-1.min-1). In all cases the uptake has, except for different Vmax values, identical kinetic and energetic properties, and is characterized by a low apparent Km value of 0.5-1.3 microM and by high substrate specificity. The transported substrate species is the deprotonated form which can also be concluded from the extremely high pH optimum of transport above pH 9. Glutamate uptake in cells grown in media with low K+ concentration is not influenced by external Na+ but is drastically stimulated by addition of K+. Stimulation by K+ could be separated into two different mechanisms. (a) Addition of K+ increases the internal pH, thereby stimulating glutamate uptake which is regulated by the internal pH in C. glutamicum. The apparent pK of the internal 'pH switch' is 6.6; below this value, uptake of glutamate is inhibited. (b) Internal K+ also directly promotes glutamate uptake. Effective uptake of glutamate can be observed only when the cytosolic K+ concentration exceeds a threshold value of about 200 mM. Stimulation of glutamate uptake by external K+ is not due to functional coupling of K+ and glutamate transport but reveals the necessity to replenish the internal K+ pool. 相似文献
104.
105.
During the ascidian sperm reaction the single large cylindrical mitochondrion which lies next to the nucleus in the head swells, becomes spherical, and migrates along the tail to be lost when it reaches the end. This sequence is initiated by eggs, egg water, high pH, low Na+, or the ionophore X537A. Accompanying the sperm reaction induced by low Na+ are H+ efflux and Ca2+ influx in a ratio of near 100:1 as determined by 45Ca2+ and atomic absorption analysis. Simultaneous pH and Ca2+ electrode measurements suggest that the movement of H+ begins 10–13 sec before the movement of Ca2+. Ca2+ uptake can be inhibited by verapamil without affecting H+ efflux or the sperm reaction. Acid release and Ca2+ uptake are proportional to the initial pH of the medium when the reaction is triggered by high pH. Acid release initiated by low Na+ is proportional to Ca2+ concentrations above 2 mM. H+ and Ca2+ movements differ in magnitude, kinetics, and inhibition by verapamil, thus suggesting that H+ is probably not exchanged for Ca2+. Instead we propose that loss of H+ triggers the uptake of Ca2+, which initiates the sperm reaction. 相似文献
106.
Eleven consecutive patients with diarrhoea from whose stools campylobacter were isolated were investigated by sigmoidoscopy and rectal biopsy. Eight had definite proctitis, and in seven biopsy specimens were abnormal with histological changes ranging from non-specific colitis to gross colitis with goblet-cell depletion and crypt-abscess formation. Nine of the patients passed blood in their stools, and in all but one abdominal pain was a feature of the illness. Severe campylobacter colitis may be clinically, sigmoidoscopically, and histologically difficult to differentiate from ulcerative colitis and is a differential diagnosis in acute colitis. 相似文献
107.
108.
109.
Quantitative zooplankton samples were obtained monthly or bi-monthly 15 times from June 1974 to May 1975 at three stations in lower Delaware Bay. Two 12-hour cruises were also conducted at one of the stations.Arthropods dominated the samples in terms of number of species and number of individuals. The number of zooplankton from surface samples ranged from 58/m3 in August to 21,092/ m3 in June, while bottom samples varied from 259/m3 in August to 30,395/ m3 in October. In general, larger concentrations of individuals were found in bottom samples.Only on three occasions did meroplankton exceed the holoplankton, and these occurred at the shallow water stations. Meroplankton comprised a larger percentage of the bottom samples than surface samples. Zoeae of Neopanope sayi and Uca sp. contributed mainly to the large proportion of meroplankton in July 1974, veligers of Mytilus edulis in January 1975, and nauplii of Balanus sp. in May 1975.Copepods were the largest component of the population throughout most of the year. At all stations and depths, Arctica tonsa dominated most of the summer samples. In the spring of 1975, A. tonsa was replaced by Centropages hamatus, Temora longicornis, and Pseudocalanus minutus.During the 12-hour cruises there were higher numbers of individuals in the bottom waters in the day with migration to surface waters in the afternoon and evening. Based on cluster analysis, five time-related assemblages were discerned: June, July–August, September–November, December, January–May. Comparison of Delaware Bay zooplankton with other estuarine systems indicated that the densities obtained locally were most similar to those reported in the York River, Virginia. 相似文献
110.
In contrast to normal mouse peritoneal macrophages, activated macrophages almost totally inhibit [3H]TdR uptake by tumor target cells 24 hr after challenge. However, when the period of observation was extended to 48 or 72 hr, renewed [3H]TdR uptake by target cells was often, but not always, observed in the presence of activated macrophages. This apparent escape of target cells from the cytostatic effects of activated macrophages was not due to a subpopulation of resistant target cells, and autoradiographic studies revealed that target cells, inhibited from incorporating [3H]TdR by activated macrophages at 24 hr, were subsequently able to renew DNA synthesis and multiply. These results suggest that in the presence of activated macrophages, the almost total cytostasis of target cells does not necessarily mean that these cells are irreversibly damaged or killed.Escape from or maintenance of cytostasis was not peculiar to any of the target cells (L cells, EMT-6, Bladder 4934) or mouse strains (SW, C57BL, BALB/c) employed nor was it consistent with any of the forms of stimulation used for obtaining activated macrophages (Toxoplasma or Besnoitia infection; C. parvum treatment). However, the results suggest that when escape of target cells from the cytostatic effects of activated macrophages occurred, it may have been due to a qualitative or quantitative inadequacy of the population of macrophages employed. 相似文献