Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “total RNA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources
PubMed1999

The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids.

OBJECTIVE: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. DESIGN: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. SETTING: Free-living individuals associated with the University of Surrey. SUBJECTS: Six male subjects with a mean (+/- sd) age of 51.2+/-3.6 y were recruited. MAJOR OUTCOME MEASURES: Pre- and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. RESULTS: Mean LPL expression values were 4.12 x 10(5) molecules of LPL mRNA per ng total RNA on the control diet and 4.60 x 10(5) molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P = 0.03, R = -0.87 and R2 = 0.75) and with the total area under the TAG-time response curve (P = 0.003, R = -0.96 and R2 = 0.92) in individuals. CONCLUSIONS: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

Adipose Tissue↗
PubMed1999

Quantitation of IGF-I, IGF-II, and multiple insulin receptor family member messenger RNAs during embryonic development in rainbow trout.

The IGF system has been shown to be important for normal embryonic growth in mice. Characterization of the IGF system in lower vertebrates is still in progress. To gain a greater understanding of the IGF system during embryonic development in teleosts, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay was developed and used to quantitate the levels of IGF-I and IGF-II mRNAs from rainbow trout embryos isolated from a staged series. The absolute number of molecules of IGF-I mRNA/microg total RNA was significantly lower than the absolute number of molecules of IGF-II mRNA/microg total RNA both during early and late embryonic development. The recent identification of multiple IGF type I receptor (rtIGFR) and insulin receptor (rtIR) cDNAs in rainbow trout has provided us with a tool to investigate the expression of these mRNAs. A relative quantitative RT-PCR assay was used to determine the steady state levels of two forms of rtIGFR and three forms of rtIR mRNAs in rainbow trout embryos. The relative levels of rtIGFR mRNAs were greater in embryos compared to adult tissues while the relative levels of rtIR mRNAs were generally lower. In a RT-PCR based assay, a differential ability to detect rtIGFR and rtIR mRNAs was shown, suggesting developmental regulation of polyadenylation. Our results suggest that IGF-II mRNA is the predominant IGF expressed in rainbow trout embryos. Our characterization of IGF ligand and receptor mRNA levels in rainbow trout embryos suggests that a functional IGF system exists during embryonic development in teleosts.

Animals↗
PubMed2004

Optimization of differential display polymerase chain reaction as a bioindicator for the cladoceran Daphnia magna.

Research on toxicant-responsive genes is providing new and important bioindicators for environmental biologists. Identifying genes whose expression is modulated by toxicant exposure provides important clues into the mechanisms underlying toxicity. In addition, toxicant-responsive genes can be developed as molecular end points that are likely to be sensitive tools for environmental assessment. Differential display polymerase chain reaction (ddPCR) is a useful approach for screening and analyzing the expression of genes. A ddPCR protocol was optimized to investigate gene expression in the cladoceran Daphnia magna. The modified protocol requires submicrogram quantities of total RNA (from <10 animals) and utilizes a sensitive fluorescent tagging system. By reverse-transcribing total RNA with arbitrary 18-nucleotide primers and PCR-amplifying the cDNA using the same arbitrary primers under low-stringency conditions, reproducible and consistent ddPCR profiles were generated. Minimal variability was introduced by reaction differences or biological variability. A trial stress (starvation) was found to generate modest differences in the ddPCR profiles. This technique promises to significantly advance knowledge regarding gene expression during toxicant insult. Furthermore, this represents the first step in the development of a novel gene fingerprinting technique that can be applied to any compound and organism of interest.

Animals↗
PubMed1995

Type I inositol-triphosphate receptor gene is alternatively spliced in human cerebral arteries.

Three distinct genes encode the three isoforms of the inositol triphosphate (IP3) receptor (type I, II and III) expressed in brain. Coupling domain of neuronal type I receptor contains a 117 nucleotide insert located between two cyclic AMP-dependent protein kinase (PKA) phosphorylation consensus sequences. By contrast, in nonneuronal tissues this insert is removed by alternative splicing. Neuronal tissue and cerebral arteries share the same embryologic origin. The present study was designed to characterize alternative splicing of the type I IP3 receptor gene in vascular tissue of human brain. Total RNA was isolated from human basilar and middle cerebral arteries and cerebellum. One microgram of total RNA was reverse transcribed. First strand cDNA was obtained and used as a template in polymerase chain reaction (PCR). PCR products were subcloned and sequenced. Specific mRNA for type I and II receptors were detected in human cerebral arteries. In vascular tissue, a short transcript was expressed indicating that the type I receptor was alternatively spliced. In contrast, only nonspliced isoform was detected in cerebellum. These results suggest that alternative splicing corresponds to differences in regulation of cerebrovascular and neuronal IP3 receptors.

Alternative Splicing↗
PubMed2001

Gene expression of insulin-like growth factor-1 and epidermal growth factor is downregulated in the heart of rats with nitrofen-induced diaphragmatic hernia.

Newborns with congenital diaphragmatic hernia (CDH) still have high mortality. Recently, a possible role of cardiac maldevelopment has been suggested. Human and experimental studies have demonstrated that heart weight is significantly reduced in the presence of CDH. Recent studies have suggested an important role for insulin-like growth factor-I (IGF-I) in the regulation of cardiac growth, structure, and function. Administration of IGF-I to normal rats has been shown to cause cardiac hypertrophy. Epidermal growth factor (EGF) plays an important role in cardiac differentiation and development. The aim of this study was to determine the gene-level expression of IGF-I and EGF in the hearts of rats with nitrofen-induced CDH using the reverse-transcription polymerase chain reaction technique (RT-PCR). CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5 of gestation (term 22 days). In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into three groups: normal controls (n = 8), nitrofen without CDH (n = 8), and nitrofen-induced CDH (n = 8). Total RNA was extracted from the hearts in each group and measured. mRNA was extracted from total RNA. RT-PCR was performed to evaluate mRNA expressions of IGF-I and EGF. Levels of mRNA were expressed as a ratio of band density divided by that of beta-actin, a housekeeping gene known to be expressed at a constant level. IGF-I mRNA expression was significantly decreased in CDH hearts (0.177 +/- 0.109) compared to controls (0.393 +/- 0.138) (P < 0.01) and nitrofen hearts without CDH (0.321 +/- 0.088) (P < 0.05). EGF mRNA expression was significantly decreased in CDH hearts (0.218 +/- 0.118) compared to controls (0.534 +/- 0.196) (P < 0.01) and nitrofen hearts without CDH (0.383 +/- 0.136) (P < 0.05). Decreased cardiac gene expression of IGF-I and EGF in the hypoplastic heart suggests that cardiac hypoplasia in nitrofen-induced rat CDH may be due to reduced synthesis of IGF-I and EGF by myocytes in the developing heart.

Animals↗
PubMed2004

Oxytocin and oxytocin-receptor mRNA expression in the human gastrointestinal tract: a polymerase chain reaction study.

BACKGROUND/AIM: Oxytocin (OT) has a wide range of effects throughout the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. So far, the few studies performed reveal no conclusive results. The aim of this study was to examine the expression of OT and OT-receptor mRNA in the human GI tract. MATERIAL AND METHODS: Full-thickness biopsies from all segments of the GI tract and the gallbladder were collected during operations at the Department of Surgery, Malmö University Hospital. Biopsies were taken and put immediately into fluid nitrogen and stored at -70 degrees C until total RNA was extracted after mechanical tissue homogenization. Subsequently, poly A(+) mRNA was isolated from the total RNA extract using an automated nucleic acid extractor and converted into single-stranded cDNA. PCR amplifications were carried out using gene-specific OT and OT-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene specific OT and OT-receptor hybridization probes. RESULTS: Expression of OT and OT-receptor mRNA was detected in nearly all segments of the GI tract analyzed. In most of the biopsy specimens analyzed, co-expression of both OT and OT-receptor mRNA appeared to take place. CONCLUSION: The present study demonstrates that OT and OT-receptor mRNAs are expressed throughout the GI tract. A possible physiological and/or pathophysiological role of OT and OT-receptor expression in the human GI tract and the cellular location of its expression remain to be shown.

Actins↗
PubMed1999

A conserved His-Asp signal response regulator-like gene in Heterosigma akashiwo chloroplasts.

Regulation of gene expression in plastids may involve molecular components conserved from cyanobacteria-like ancestors. Among prokaryotes, genes are commonly regulated at the transcriptional level by 'two-component' or 'His-Asp' signal transducers, consisting of a 'sensor kinase', which autophosphorylates at a conserved histidine residue, and a cognate response regulator, which is phosphorylated by the sensor kinase at a conserved aspartate residue. A putative His-Asp response regulator gene (trg1: transcriptional regulatory gene 1) has been identified in the estuarine raphidophytic alga Heterosigma akashiwo. The chloroplast-encoded trg1 is 693 bp in length, contains no introns, and yields a conceptual translation product of 231 amino acids, with a predicted mass of 27 kDa. Homology searches suggest that Heterosigma trgl has an omnpR-like identity within the DNA-binding His-Asp family of response regulators. trg1 contains both the phosphorylation and DNA-binding domains which are present in prokaryote response regulators. Quantitative competitive RT-PCR showed that Heterosigma trg1 is expressed at low levels (5 microg per g total RNA). In contrast, psbA (a photosystem II component) transcript is abundant (60 mg per g total RNA). Cell cycle analysis showed that psbA abundance oscillates in response to light but trg1 mRNA levels are invariant. We hypothesize that a His-Asp phosphorelay mechanism may affect chloroplast genome transcription in a manner similar to bacterial signal transduction pathways in which 'sensor kinase' and cognate 'response regulator' proteins interact.

Algal Proteins↗
PubMed1996

Alpha1,3-L-fucosyltransferase expression in developing human myeloid cells. Antigenic, enzymatic, and mRNA analyses.

In an attempt to correlate the cell surface expression of Lex and sialyl-Lex structures in immature and mature myeloid cells with the genes expressing alpha1,3-fucosyltransferase(s) we have examined: 1) the properties of the cellular alpha1,3-fucosyltransferases and the mRNA transcripts corresponding to the five cloned genes, Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, and Fuc-TVII, in mature granulocytes and in the myeloid cell line HL-60, before and after dimethyl sulfoxide-induced differentiation and 2) the properties of the alpha1,3-fucosyltransferases expressed in COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNAs. The previously shown increase in cell surface expression of sialyl-Lex on differentiation of HL-60 cells (Skacel P. O., Edwards A. J., Harrison C. T., and Watkins W. M. (1991) Blood 78, 1452-1460) is accompanied by a sharp fall in expression of Fuc-TIV mRNA and a persistence of expression of Fuc-TVII mRNA. The properties of the alpha1,3-fucosyltransferase expressed in COS-7 cells transfected with Fuc-TIV are consistent with this being the major gene responsible for the expression of Lex in the immature myeloid cells. In Northern blot analyses, no transcripts of Fuc-TIII, Fuc-TV, or Fuc-TVI were detected in total RNA from mature granulocytes or mRNA from HL-60 cells before or after differentiation. In total RNA from mature granulocytes, Fuc-TIV transcripts were only faintly visible, whereas Fuc-TVII transcripts were quite definitely expressed. The specificity properties of Fuc-TVII expressed in COS-7 cells are consistent with this gene being the major candidate alpha1, 3-fucosyltransferase controlling the expression of sialyl-Lex on mature cells. However, Lex continues to be expressed on the surface of mature granulocytes and cell extracts retain the capacity to transfer fucose to non-sialylated acceptor substrates. The question therefore remains as to whether these properties result from the weakly expressed Fuc-TIV gene or whether another alpha1, 3-fucosyltransferase gene remains to be identified.

Animals↗
PubMed1998

Quantification of mRNA levels of endothelin receptor subtypes and preproEndothelin-1 in renal needle biopsies by competitive reverse transcriptase polymerase chain reaction.

The vasoconstrictive peptide endothelin-1 (ET-1) is an autocrine/paracrine peptide of putative pathophysiological importance in renal transplant medicine. The aim of the present study was to develop a method for analysis of gene expression of the renal endothelin system in humans. Only small amounts of tissue are available from renal cortical needle biopsies. Thus, in the present study we developed a quantitative assay based on the competitive reverse transcriptase polymerase chain reaction (RT-PCR) technology. We quantified endothelin A (ET(A)) and B (ET(B)) receptor subtype mRNAs and preproET-1 mRNA levels in renal cortex biopsies obtained before nephrectomy of healthy kidney donors. Mean (+/- SEM) mRNA levels of the ET(A) and ET(B) receptor subtypes in 26 living donors were 212 +/- 23 and 368 +/- 56 amol/microg total RNA, respectively. The preproET-1 mRNA level in 19 living donors was 213 +/- 28 amol/microg total RNA. The inter-assay coefficient of variation (CV) for the assay was 10%; the intra-assay CV was 6-13%. The competitive RT-PCR assay described provides an accurate tool for gene expression investigation of the human endothelin system in renal cortical needle biopsies.

Adult↗
PubMed2002

Quantification of mRNA for endothelial NO synthase in mouse blood vessels by real-time polymerase chain reaction.

The mouse is useful in studies of vascular biology because of its well-defined genetics and because the mouse genome can be manipulated. However, because only small amounts of mRNA can be extracted from blood vessels, the quantification of gene expression in individual mice is difficult. Endothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth. In addition, there appear to be sex differences in the production of NO under basal conditions in mouse aortas. The goals of this study were to develop a real-time polymerase chain reaction (PCR) method to quantify eNOS mRNA in blood vessels from mice and to examine eNOS mRNA levels in vessels from male and female mice. Blood vessels were isolated from C57BL/6 mice. Total RNA from individual mice was isolated and reverse-transcribed. The number of molecules of eNOS mRNA (after reverse transcription) was determined against cDNA standards, with 18S rRNA used as a control for RNA input and reverse-transcription efficiency. When expressed as copy numbers per nanogram of total RNA or as the ratio of eNOS to 18S rRNA, eNOS mRNA was lower in the aortas of female mice than in those of male mice at 7 to 9 months of age. In contrast, no difference in eNOS mRNA was found in the aortas of 2-month-old mice. In addition, eNOS mRNA levels were similar in the carotid, cerebral, and coronary arteries. These findings provide the first quantitative measurements of eNOS mRNA by using real-time PCR in the vessels of mice and suggest age- and sex-related differences in the basal levels of eNOS mRNA in mice. In addition, the eNOS region that was used for real-time PCR was amplified and sequenced for monkeys and other species. With modifications, this region may be used to design real-time PCR for eNOS in other species.

Animals↗
PubMed1990

Effect of dietary protein on rat renin and angiotensinogen gene expression.

Plasma renin activity varies with the level of dietary protein, being higher on a high protein diet. To explore the molecular mechanisms underlying this relationship we first examined the effect of dietary protein on renin and angiotensinogen gene expression at the level of steady state mRNA in male Sprague-Dawley rats. Renal renin mRNA was higher on a 50% (high) compared to a 6% (low) protein diet both 3 d (9.4 +/- 1.1 vs. 5.3 +/- 0.4 pg/micrograms of total RNA; P less than 0.02) and 21 d (6.8 +/- 1.0 vs. 3.5 +/- 0.4 pg/micrograms of total RNA; P less than 0.02) after dietary change. No change occurred in either renal or liver angiotensinogen mRNA. When three levels of dietary protein were examined, renal renin mRNA was elevated on a 50% and lowered on a 6% protein diet compared to a more standard 20% protein diet. Kidney weights and renal protein, RNA, and RNA/DNA increased with the level of dietary protein reflecting protein-induced renal hypertrophy. Uninephrectomy resulted in no change in renin mRNA compared to sham operation (3.7 +/- 0.1 vs. 3.4 +/- 0.1 pg/micrograms RNA; P = NS) despite renal growth in the uninephrectomy group implicating dietary protein and not hypertrophy as the major factor for stimulating renin mRNA. In conclusion, the level of dietary protein is a novel and specific stimulus for changes in renal renin mRNA. The increased plasma renin activity on a high protein diet is due at least in part to increased renin synthesis.

Angiotensinogen↗
PubMed1997

Detection of a tyrosine-phosphorylated form of cyclin A during liver regeneration.

Cyclin A functions in both the S and G2-M phases of the cell cycle. The expression of cyclin A during liver regeneration was compared with that of cyclin B1 and p34cdc2. Liver regeneration was followed at 2-h intervals from 12 to 48 h after partial hepatectomy (PH). Immunohistochemical staining using proliferating cell nuclear antigen revealed DNA synthesis peaks at 18 h after PH. The most intense nuclear staining of hepatocytes with cyclins A and B1 and p34cdc2 antibodies occurred at 26 h post-PH, which corresponds with the onset of mitosis. Quantitative mRNA expression of cyclins A and B1 and p34cdc2 was determined by competitive reverse transcription-PCR. Construction of mRNA internal standards and coamplification during reverse transcription-PCR allowed quantitation of all three cell cycle genes. At 24 h post-PH, cyclin A mRNA levels were approximately 5 fg/100 ng total RNA. In contrast, cyclin B1 and p34cdc2 levels were 20-fold higher, 100 fg/100 ng total RNA. Cyclin B1 and p34cdc2 mRNA levels showed two peaks, at 26 and 38-44 h post-PH, whereas the levels of cyclin A were constant during this interval. Immunoblots revealed the presence of cyclin A in normal liver, and significant amounts were present as early as 12 h post-PH. At 26 h post-PH, tyrosine-phosphorylated forms of cyclin A were detected. Cyclin B1 and p34cdc2 protein were not present until 22-24 h post-PH, and two peaks were observed, at 26 and 38-44 h, coinciding with the mRNA pattern. Histone H1 kinase activity was associated with the two peaks of cyclin B1 and p34cdc2 expression. The unique pattern of cyclin A expression and detection of tyrosine-phosphorylated forms suggest a different mechanism for the regulation of cyclin A during liver regeneration.

Animals↗
PubMed1978

Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells.

Glucocorticoid hormone treatment of GR cells, a cultured line derived from mouse mammary tumor tissue, selectively stimulates the rate of transcription of integrated proviral genes specifying mammary tumor virus (MTV). We have incubated isolated nuclei from these cells under conditions in which all three endogenous RNA polymerases appear to be active. RNA synthesized in vitro is distinguished from preexisting nuclear RNA by labeling the in vitro products with [3H]CTP, and the level of MTV RNA synthesis is measured by molecular hybridization with unlabeled viral DNA. Synthesis requires the addition of nucleoside triphosphates, and is inhibited by actinomycin D. Pretreatment of GR cells with dexamethasone, a synthetic glucocorticoid, has no significant effect on the amount of total RNA synthesis in isolated nuclei. In contrast, synthesis of MTR RNA is stimulated 10-20-fold in nuclei from dexamethasone-treated cells relative to untreated control nuclei; the sensitivity of in vitro viral RNA synthesis to inhibition by alpha-amanitin suggests that it is carried out exclusively by RNA polymerase II. The fraction of total RNA synthesis which is viral specific (about 0.2-0.4% in nuclei from dexamethasone-treated cells and 0.01-0.03% in controls) is similar to that detected in pulse labeled RNA from whole GR cells in culture. Our procedures for labeling and hybridization of RNA appear to avoid artifacts recently noted in other in vitro transcription systems.

Amanitins↗
PubMed1987

Messenger RNA content and complexity in normal and overloaded rat heart.

Cardiac overload was studied in rats by abdominal aortic constriction, which increased the left ventricular weight by 59% after 12 days. During the transition, which precedes the compensatory hypertrophy, both total RNA and poly(A)-containing messenger RNA increased. The concentration of these polynucleotides peaked by day 4 after constriction, rising from 1.27 +/- 0.3 to 1.88 +/- 0.2 mg g-1 fresh weight for total RNA, and from 38 +/- 24 to 62 +/- 12 micrograms g-1 fresh weight for poly(A)-containing RNA, and returned to normal by day 12. However, the total amount per ventricle of both RNAs remained high. Poly(A)-containing RNA prepared from normal heart was hybridized to its cDNA copy. These results were expressed as the percentage of hybridization vs. the log 10 of the product of the poly(A)-containing RNA concentration and the time (Rot), and in computer analysis were described by division into three different frequency components. In normal hearts, the Rot 1/2 values of these components were, respectively, 3.98 X 10(-3), 0.338 and 21.380 mol.s.1(-1), which correspond to 2-3, 240 and 12,200 different sequences that were copied 22,000-33,000, 310 and 5 times, respectively. Four and 30 days after banding there was a harmonious enhancement of the number of the copies without any change in the number of different sequences, and the three different hybridization curves were superimposed. In conclusion, cardiac overload raises the poly(A)-containing RNA concentration, probably by stimulating transcription, but no major changes occur in any of the frequency classes.

Animals↗
PubMed1988

Isolation and in vitro translation of mRNA from rat peritoneal mast cells and rat basophilic leukemia cells.

In the absence of any specific literature on the isolation of RNA from mast cells, our initial attempts established that unusual measures would be needed to prepare acceptable yields of high quality RNA from peritoneal mast cells of normal adult rats. Accordingly, we developed procedures for the isolation and characterization of RNA from rat peritoneal mast cells (PMC) and basophilic leukemia cells (RBL). The significant components of the procedures include: separation and removal of mast cell granules to minimize contamination of RNA with proteins and proteoglycans; use of bentonite in phenol extractions; and repetition of extractions and precipitation. The amounts of total RNA extracted from PMC were about 15% of those from RBL, although the percentage mRNA of total RNA in PMC and RBL was similar (1.8 and 2.0%). Ribosomal RNA banding patterns in agarose gel electrophoresis and in vitro translation experiments indicate that the isolated RNA can be employed for analysis of molecular mechanisms of mast cell function and heterogeneity.

Animals↗
PubMed2002

Induction of arginase II in livers of bile duct-ligated rats.

Nitric oxide (NO) has been implicated in playing a role in liver cirrhosis, but the regulatory mechanisms are still unclear. As arginase shares a common substrate with NO synthase (NOS), the aim of this study was to investigate the expression of arginase I and II in cirrhotic liver. Liver cirrhosis was induced in rats by chronic bile duct ligation (BDL). Controls were sham-operated. Competitive polymerase chain reaction was performed to assay the expression of messenger RNA of arginase I and II. Protein expression was detected by immunohistochemistry and western-blotting. The level of arginine in plasma was lower in BDL rats, while the ornithine level in plasma was correspondingly higher (r= -0.96, P<0.0001). Arginase I messenger RNA was reduced significantly in BDL rats (3.34+/-0.32 vs. 1.32+/-0.21 x 10(4) attomole/microg of total RNA, sham vs. BDL, P<0.001), as well as arginase I protein. In contrast, arginase II mRNA was induced in the livers of BDL rats, with negligible expression in controls (0.35+/-0.11 vs. 3.64+/-0.54 attomole/microg of total RNA, sham vs. BDL, P<0.001). Arginase II protein was localized in some hepatocytes and hyperplastic bile ductular epithelial cells of cirrhotic livers but not in control livers. In conclusion, arginase II was induced in BDL livers, while the expression of arginase I was down-regulated. These data suggest that arginase I and II are regulated differently and may have different functions in the livers of BDL rats. Reduction of arginase I in BDL livers may be responsible for the lowering of arginine levels in the plasma, while induction of arginase II could be important in regulating NO synthesis as well as other important mechanisms involved in liver cirrhosis.

Animals↗
PubMed1997

Differential gene expression of steroid 5 alpha-reductase 2 in core needle biopsies from malignant and benign prostatic tissue.

Androgens are implicated in the development of prostate cancer (CAP) and benign prostate hyperplasia. The conversion of testosterone to the more potent metabolite dihydrotestosterone by prostatespecific steroid 5 alpha-reductase type 2 (5 alpha-red2) is a key mechanism in the action of androgens in the prostate and is important in the promotion and progression of prostate diseases. Manipulation of the turnover of androgens is thus fundamental in the pharmacological treatment strategy. We have developed a sensitive solution hybridization method for quantification of the gene expression of 5 alpha-red2 in core needle biopsies of the prostate. The 5 alpha-red2-specific messenger RNA (mRNA) levels were measured in 50 human prostate transrectal ultrasound-guided core biopsies obtained from 31 outpatients (median age 72, range 67-88 yr) undergoing biopsy for diagnostic purposes. Significant differences were observed in the gene expression of 5 alpha-red2 between cancerous and noncancerous tissue. In the 14 biopsies judged cancerous, the median 5 alpha-red mRNA levels were 3.5 amol/ng total RNA compared with 12.0 amol/ng total RNA in the biopsies showing no cancer (P = 0.0018). The median 5 alpha-red2 mRNA level in noncancerous tissue was thus 3.4 times higher than in the cancerous specimens.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Time course and cellular localization of lipopolysaccharide-induced inducible nitric oxide synthase messenger RNA expression in the rat in vivo.

OBJECTIVES: The role of nitric oxide in mediating the early phase of hypotension and cardiovascular hyporeactivity in sepsis, and the cellular sources of inducible nitric oxide synthase expression under in vivo conditions, remain unclear. The objective of this study was to elucidate the time course and cellular location of inducible nitric oxide synthase messenger RNA (mRNA) expression in rats treated with lipopolysaccharide in vivo. DESIGN: Prospective, placebo-controlled, laboratory study. SETTING: Experimental laboratory of a postgraduate medical research institution. SUBJECTS: Normal, anesthetized rats. INTERVENTIONS: Animals were treated with lipopolysaccharide (15 mg/kg i.p.), saline (1 mL/kg i.p.), or lipopolysaccharide plus dexamethasone (3 mg/kg i.p., 50 mins before lipopolysaccharide administration) in vivo 4 hrs before the removal of tissues. MEASUREMENTS AND MAIN RESULTS: Total RNA and mRNA were extracted. Total RNA was used for reverse transcription polymerase chain reaction. Poly-(A)+ mRNA was isolated for Northern blot analysis. Cryostat sections of heart and lung were prepared for in situ hybridization to elucidate the cellular location of the inducible nitric oxide synthase gene. Hearts, lungs, aortae, and pulmonary arteries from lipopolysaccharide-treated animals expressed inducible nitric oxide synthase mRNA, which was markedly inhibited by pretreatment with dexamethasone (except in aorta). The threshold time point for inducible nitric oxide synthase mRNA induction was between 20 and 40 mins after lipopolysaccharide administration. The quantity of mRNA increased progressively thereafter, reaching a plateau between 4 and 8 hrs and decreasing markedly by 24 hrs. These findings are consistent with the time course of lipopolysaccharide-induced hypotension and cardiovascular hyporeactivity in animal models of septic shock and in man. Inducible nitric oxide synthase mRNA was mainly detected in vascular and airway smooth muscle cells, cardiac myocytes, pneumocytes, and infiltrated inflammatory cells, but was also identified in airway epithelial and vascular endothelial cells. Vascular smooth muscle represented the predominant cell type expressing inducible nitric oxide synthase under these conditions. CONCLUSIONS: These results provide further molecular evidence for the involvement of nitric oxide in the early phase of hypotension and cardiovascular hyporeactivity seen in septic shock. They also suggest that nitric oxide derived from vascular smooth muscle cells may contribute significantly to this hypotension and cardiovascular reactivity.

Animals↗