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Mechanism of maintenance of liver-specific functions by DMSO in cultured rat hepatocytes 总被引:2,自引:0,他引:2
The present study was undertaken to investigate the mechanism by which dimethylsulfoxide (DMSO) exerts its protective action on cytochrome P450-dependent activities and differentiation in cultured rat hepatocytes. Loss of cytochrome P450 is associated with a shortage of heme and reduced activity of delta-aminolaevulinic acid dehydratase: the addition of DMSO, which induces this enzyme in human hepatoma cells, is not able to affect it in hepatocytes in primary culture. DMSO is a strong scavenger of hydroxyl radicals and may destroy the reactive oxygen species formed under conventional culture conditions (i.e., 95% air and 5% CO2). In fact other powerful scavengers of oxygen radicals like dimethylthiourea, desferal, and catalase itself maintain higher levels of cytochrome P450 and higher activities of 7-ethoxycoumarin O-deethylase during 3 days of culture. DMSO and the other scavengers are also able to retain features of the morphological and biochemical differentiation of hepatocytes such as the ability to induce tyrosine aminotransferase activity in response to glucocorticoids. 相似文献
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We have optimized a liposome-based transfection method that mediated highly efficient stable expression of foreign genes in hepatocytes. Moreover, we have observed that the metallothionein 1 promoter in the bovine papilloma virus-based expression vector drove the highest expression of foreign genes in hepatocytes as compared with the cytomegalovirus and the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoters in the pcDNA 3-based expression vector. The cytomegalovirus promoter failed to yield significant expression in these cells. Furthermore, expression of foreign genes persisted up to at least 15 passages when expression was under the control of either the EF-1alpha or the metallothionein 1 promoter. Thus, these two promoters led to comparable stability of foreign genes in hepatocytes, with the metallothionein 1 promoter yielding a higher level of expression of foreign genes in these cells. 相似文献
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Cytoplasmic processing events in the poly(A) region of mRNA fromPhysarum polycephalum are reviewed. Two classes of poly-containing RNA [poly(A)+ RNA] exist in the cytoplasm. One contains very short poly(A) sequences, averaging about 15 adenylate residues, while the other contains relatively long poly(A) sequences, averaging about 60 residues. Molecules with short poly(A) sequences are found exclusively in the polysomes while those with long poly(A) sequences are restricted to the free cytoplasmic mRNP. Since proteins are associated with only the long poly(A) sequences the poly(A) · protein complex is also restricted to the free mRNP. The long poly(A) sequences are relatively short-lived. They are degraded by two distinct processes, a shortening process in which 15–20 residues are gradually removed and a turnover process in which long poly(A) tracts are rapidly converted to the short sequences. This process, along with the dissociation of the poly(A) · protein complex, occurs when poly(A)+ RNA molecules located in free mRNP are transferred to the polysomes. Poly(A) · protein complex dissociation appears to preceed poly(A) turnover during translational selection. The significance of these processing events in relation to mRNA maturation is discussed. 相似文献
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Gaoxiong Wang Youshi Zheng Yingchao Wang Zhixiong Cai Naishun Liao Jingfeng Liu Wenmin Zhang 《Cytotechnology》2018,70(4):1279-1290
Although hepatocyte transplantation and bioartificial liver support system provide new promising opportunities for those patients waiting for liver transplantation, hepatocytes are easily losing liver-specific functions by using the common in vitro cultured methods. The co-culture strategies with mimicking the in vivo microenvironment would facilitate the maintenance of liver-specific functions of hepatocytes. Considering that hepatocytes and endothelial cells (ECs) account for 80–90% of total cell populations in the liver, hepatocytes and ECs were directly co-cultured with hepatic stellate cells (HSCs) or adipose tissue-derived stem cells (ADSCs) at a ratio of 700:150:3 or 14:3:3 in the present study, and the liver-specific functions were carefully analyzed. Our results showed that the two co-culture systems presented the enhanced liver-specific functions through promoting secretion of urea and ALB and increasing the expressions of ALB, CYP3A4 and HNF4α, and the vessel-like structure in the co-culture system consisted of hepatocytes, ECs and ADSCs. Hence, our results suggested that the directly co-culture of hepatocytes and ECs with HSCs or ADSCs could significantly improve liver-specific functions of hepatocytes, and the co-culture system could further promote angiogenesis of ECs at a later stage. Therefore, this study provides potential interesting in vitro strategies for enhancing liver-specific functions of hepatocytes. 相似文献
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The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids. 相似文献
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Cavallini Giorgio Bovo Paolo Bianchini Ercolina Carsana Antonella Merola Marcello Sgarbi Daniela Palmieri Marta 《Molecular and cellular biochemistry》1998,185(1-2):147-152
Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous tissue from pancreatic cancer subjects. However, about 70% of the pancreatic cancer subjects showed lithostathine and chymotrypsinogen mRNA levels comparable to those of chronic pancreatitis patients. These results indicate that the decrease in the level of mRNA is not specific to lithostathine and it is unrelated to the presence of pancreatic stones. 相似文献
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The RNA contents in rat and human liver cells was measured using the scanning absorbtion photometric method after gallocyanin-chromalum staining. The RNA content was shown to increase proportionally with the increase of genome numbers in hepatocytes. 相似文献
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We describe the isolation of a cDNA clone of β1,3-glucanase mRNA from Nicotiana tabacum L. cv. `Havana 425' and its use to measure the kinetics of mRNA accumulation in cultured tobacco tissues treated with the plant hormones auxin and cytokinin. Northern blot analysis showed that the tissues contain a single ˜1.6 kb-sized β1,3-glucanase mRNA. The levels of β1,3-glucanase and β1,3-glucanase mRNA increase by up to seven- and 20-fold, respectively, over a 7-day period in tissues subcultured on hormone-free medium and medium containing auxin or cytokinin added separately. Over the same interval of time, the content of both the enzyme and its mRNA remains at a constant low level in tissues subcultured on medium containing both auxin and cytokinin. The results show that auxin and cytokinin block β1,3-glucanase production at the level of the mRNA. 相似文献
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Modulation of thyroglobulin messenger RNA level by thyrotropin in cultured thyroid cells. 总被引:3,自引:0,他引:3
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To examine the influence of thyrotropin (TSH) on the thyroglobulin (Tgb) mRNA content, the latter was evaluated in the cytoplasm of hog thyroid cells cultured in the absence (control cells) or presence of TSH. The Tgb mRNA levels were determined by, (i) kinetics of hybridization to sheep Tgb cDNA, (ii) capacity of coding for peptides immunologically related to Tgb in reticulocyte lysate. In cells cultured for 4 days in the absence of TSH, the content of Tgb mRNA sequences decreased to 30% of its initial value and the messenger activity to 15%. Conversely, TSH maintained the initial Tgb mRNA level in cells cultured in its presence, and TSH concentrations 50 micronU/ml or 5 mU/ml gave identical results. At each period tested poly (A) content was the same in TSH-treated and control cells. When TSH was added to media after 4 or 8 days culture without TSH, the Tgb mRNA level was partially restored. These results suggest that TSH exerts a positive control on Tgb gene expression through modulation of Tgb mRNA content of thyroid cells. 相似文献
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Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells 总被引:2,自引:0,他引:2
P S LaPolt D Soto J G Su C A Campen J Vaughan W Vale A J Hsueh 《Molecular endocrinology (Baltimore, Md.)》1989,3(10):1666-1673
Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Immunocytochemical localization of liver-specific proteins in pancreatic hepatocytes of rat 总被引:3,自引:0,他引:3
Hepatocytes are induced in the pancreas of rats maintained first on a copper-deficient diet for 8 weeks and then on normal rat chow. These cells are morphologically identical to parenchymal cells of the liver. These hepatocytes contain two liver-specific proteins: carbamyl phosphate synthetase I, a mitochondrial matrix protein that participates in the conversion of ammonia to carbamyl phosphate; and urate oxidase, an enzyme that catalyzes the oxidation of uric acid to allantoin. In addition, we also present evidence indicating that dietary administration of ciprofibrate induces peroxisomal beta-oxidation pathway enzymes, while the levels of catalase are unaltered in pancreatic hepatocytes. These observations along with the previously published results further establish the identity of pancreatic hepatocytes to parenchymal cells of liver and clearly indicate that transdifferentiation of pancreatic cells to hepatocytes is associated with activation of several liver-specific genes. 相似文献
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From a DNA-directed cell-free system, functional gal mRNA is obtained which directs the cell-free synthesis of the three galactose enzymes of Escherichia coli. A substantial fraction of this gal mRNA has the properties of a polycistronic messenger. Exposure to elevated temperatures in the presence or absence of magnesium ion results in pronounced changes of the capacity of this mRNA to give rise to the synthesis of the three enzymes. Depending on the conditions of the pre-treatment, the absolute amounts as well as the ratio of the three gene products synthesized can be changed. The different forms of gal messenger so obtained also exhibit different susceptibilities towards functional inactivation during the enzyme synthesis reaction. As the changes in template activity are reversible, it is concluded that the different treatments cause reversible transitions between different conformations of the gal mRNA. 相似文献