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PubMed1978

Variability in the amount of beta-globin mRNA in beta0 thalassemia.

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.

Blood Cells↗

PubMed1985

Characterization of infectious hematopoietic necrosis virus mRNA species reveals a nonvirion rhabdovirus protein.

The genome RNA and six mRNA species of infectious hematopoietic necrosis virus were analyzed by denaturing gel electrophoresis. The following molecular weights were determined: genome RNA, 3.7 X 10(6); mRNA 1, 2.26 X 10(6); mRNA 2, 5.63 X 10(5); mRNA 3, 4.84 X 10(5); mRNA 4 (containing two different mRNA species), 3.00 X 10(5); and mRNA 5, 1.95 X 10(5). Densitometer analyses of gels were used to calculate the molar ratios of the intracellular mRNA species: mRNA 1, 0.02; mRNA 2, 0.49; mRNA 3, 1.0; mRNA 4, 2.52; and mRNA 5, 0.41. Hybrid selection studies determined the mRNA coding assignments as follows: mRNA 1 encodes the viral polymerase, L; mRNA 2 encodes the glycoprotein, G; mRNA 3 encodes the nucleocapsid protein, N; mRNA 4 is composed of two comigrating mRNA species which encode the matrix proteins, M1 and M2; and mRNA 5 encodes a previously unrecognized viral protein which is induced in infected cells but is not present in mature virions. This nonvirion protein has been designated the NV protein.

Animals↗

PubMed1983

Characterization of cytochrome P2-450 (20-S) mRNA. Association with the P1-450 genomic gene and differential response to the inducers 3-methylcholanthrene and isosafrole.

Mouse liver cytochrome P2-450 is defined as the major isosafrole-inducible form of P-450 which is most specific for isosafrole metabolism. lambda AhP-1 represents a 15.5 X 10(3)-base-pair segment of mouse genomic DNA having the cytochrome P1-450 gene (approximately equal to 4600 base pairs) located in the middle portion. Using various subclones as probes, we investigated the differential expression of P1-450 mRNA and P2-450 mRNA induction as a function of association with the Ah locus, 3-methylcholanthrene or isosafrole dosage, tissue specificity, and developmental age. Both P1-450 (23-S) mRNA and P2-450 (20-S) mRNA induction processes are regulated by the Ah receptor. P2-450 mRNA is about 10-fold more sensitive than P1-450 mRNA to induction by either 3-methylcholanthrene or isosafrole. Phenobarbital pretreatment has no effect at all on either P1-450 mRNA or P2-450 mRNA. Whereas both P1-450 mRNA and P2-450 mRNA are induced by 3-methylcholanthrene in C57BL/6N liver, P1-450 (23-S) mRNA but not P2-450 (20-S) mRNA is induced by 3-methylcholanthrene in C57BL/6N kidney. P1-450 mRNA induction by 3-methylcholanthrene is measurable in C57BL/6N liver at day 15 of gestation, and the expression becomes enhanced with increasing age. P2-450 mRNA induction by 3-methylcholanthrene in C57BL/6N liver appears about 7 days later during development than 3-methylcholanthrene-inducible P1-450 mRNA. Both 3-methylcholanthrene-induced P1-450 mRNA and P2-450 mRNA are detectable in DBA/2N liver; their appearance is later in development, however, and at lower concentrations than that seen with C57BL/6N liver. P1-450 (23-S) mRNA and P2-450 (20-S) mRNA appear to hybridize to a common 5' fragment of the P1-450 gene.

Animals↗

PubMed1987

Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle.

In muscle cells two populations of mRNA are present in the cytoplasm. The majority of mRNA is associated with ribosomes and active in protein synthesis. A small population of cytoplasmic mRNA occur as free mRNA-protein complex and is not associated with ribosomes. This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library. Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA. One of these clones was extensively studied. The DNA from a recombinant plasmid D12 hybridized to a 1.3-kb poly(A)-rich mRNA. In proliferating myoblast cells, the 1.3-kb mRNA was more abundant in the polysomal fraction and mostly free in the non-dividing myotubes. In contrast to this mRNA, 90% of alpha and beta actin mRNAs were translated in both myoblasts and myotubes. Further analysis of distribution of the 1.3-kb RNA in the polysomal (active) and free (repressed) fractions in fusion-arrested postmitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA. Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA. The presence of this mRNA was not, however, limited to muscle cells. This mRNA was also present in the repressed state in rat liver and kidney cells. These results, therefore, suggest that the 1.3-kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non-dividing cells. Messenger RNA homologous to the 600-base-pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes. Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell-free system synthesized a 40-kDa polypeptide. These results suggest that the repressed population of 1.3-kb mRNA can be translated in vitro. The hybridization pattern of DNA from the recombinant plasmid D12 with rat genomic DNA suggested that the 1.3-kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome.

Actins↗

PubMed1998

Luteal expression of steroidogenic factor-1 mRNA during the estrous cycle and in response to luteotropic and luteolytic stimuli in ewes.

Steroidogenic factor-1 (SF-1) is a transcription factor involved in regulating basal and/or cAMP-induced increases in expression of several components of the steroidogenic pathway, including cytochrome P450 side-chain cleavage (P450scc), steroidogenic acute regulatory protein (StAR), and 3beta-hydroxysteroid dehydrogenase/delta5, delta4 isomerase (3beta-HSD). In experiment 1, on days 3, 6, 9, 12, and 15 of the estrous cycle, steady-state concentrations (fmol/microg poly A+ RNA) of SF-1 mRNA in luteal tissue were 0.09 +/- 0.01, 0.17 +/- 0.01, 0.24 +/- 0.03, 0.30 +/- 0.09, and 0.20 +/- 0.05, respectively (estrus = day 0; n = 4/d). Concentrations of SF-1 mRNA increased (p < 0.05) between days 3 and 12, but were not different among the other days of the estrous cycle. Luteal concentrations of SF-1 mRNA and concentrations of progesterone in sera were highly correlated (p < 0.01; r = 0.72). In experiment 2, ewes on days 11 or 12 of the estrous cycle were injected with 25 mg prostaglandin F2alpha (PGF2alpha) into the jugular vein followed by an injection of 10 mg PGF2alpha i.m. 2 h later. Corpora lutea were collected 4, 12, and 24 h after the first injection of PGF2alpha (n = 4-5 ewes/time). Control luteal tissue was collected from ewes on days 11-13 of the estrous cycle, which had not been injected (n = 4) or had been injected with saline 24 h previously (n = 4). Steady-state concentrations of SF-1 mRNA had decreased (p < 0.05) to 48% of control values by 4 h after injection, and remained low at 12 and 24 h. In experiment 3, ewes on days 9-12 of the estrous cycle were administered PGF2alpha (1 micromol), phorbol 12-myristate 13-acetate (PMA; 2 micromol), luteinizing hormone (LH; 20 microg), forskolin (50 microg), or vehicle (1 mL saline) directly into the ovarian artery. Corpora lutea were collected 0 (noninfused) 4, 12, or 24 h later (n = 3-4 animals/treatment/time) for quantification of SF-1 mRNA. Steady-state concentrations of mRNA encoding SF-1 were not affected by infusion of PGF2alpha or PMA, although concentrations of mRNA encoding StAR and 3beta-HSD were decreased (p < 0.05) by these treatments. Concentrations of mRNA encoding SF-1 were increased (p < 0.05) to 157 and 149% of control values by LH and forskolin, respectively, 12 h following infusion and returned to control values by 24 h following either treatment. In contrast, infusion of LH or forskolin did not change concentrations of mRNA encoding StAR, P450scc, or 3beta-HSD. In summary, during the estrous cycle, the pattern of expression of SF-1 mRNA was similar to the pattern of concentrations of progesterone in serum and expression of mRNA encoding P450scc, but differed from that previously shown for 3beta-HSD and StAR mRNA. The effects of administration of PGF2alpha on concentrations of SF-1 mRNA appeared to be dose-dependent. However, acute effects of PGF2alpha on mRNA encoding 3beta-HSD and StAR were observed when concentrations of mRNA encoding SF-1 were not influenced. In addition, although LH or forskolin increased luteal SF-1 mRNA 12 h following infusion, no increases in mRNA encoding StAR, P450scc, or 3beta-HSD were observed. Thus, during the midluteal phase of the estrous cycle, neither luteotropic nor luteolytic hormones appear to coordinately regulate mRNA encoding SF-1 and mRNA encoding StAR, P450scc, or 3beta-HSD.

3-Hydroxysteroid Dehydrogenases↗

PubMed2001

Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA.

During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.

Adult↗

PubMed1993

p21X mRNA is expressed as a singly spliced pX transcript from defective provirus genomes having a partial deletion of the pol-env region in human T-cell leukemia virus type 1-infected cells.

In addition to the three typical transcripts such as genomic/gag-pol mRNA, env mRNA and tax/rex mRNA, we previously found the singly spliced pX mRNA, termed p21X mRNA, responsible for producing the p21X protein in human T-cell leukemia virus type 1 (HTLV-1)-infected cells. Our finding of the p21X mRNA being constitutively expressed in the fresh peripheral blood mononuclear cells (PBMCs) from patients with ATL has suggested that the expression mechanism is quite different from that of the others. In this paper, the expression mechanism of p21X mRNA was investigated by analyzing the organization of the proviral genomes present in the representative HTLV-1-infected cell lines which are positive or negative for the expression of p21X mRNA. Southern and PCR analyses show that most of the analyzed cell lines contain both one complete and one defective genome each. However, one cell line without the p21X mRNA expression, C91/PL, contains only the complete genome, suggesting that the complete HTLV-1 has no ability to express p21X mRNA in spite of having the ability to produce the infectious virus. The defective genomes of the p21X mRNA positive cell lines, MT-2 and H582, have a large deletion of the entire pol and parts of the gag and env regions including the common domain of the second exon of the doubly spliced tax/rex mRNA, while another defective genome of the p21X mRNA negative cell line, MT-1, has a deletion within the gag-pol gene. We show that these defective genomes have the ability to express their distinct, defective genomic mRNA, suggesting they are active. The defective genomic mRNAs in MT-2 and H582 cells retain the first splice donor and the second splice acceptor sites, suggesting the possibility of synthesizing p21X mRNA by splicing singly with these sites. These findings assume that defective HTLV-1 genomes deleting the second exon region acquire the ability to express p21X mRNA but no ability to express tax/rex mRNA. Such a deletion may explain the difference between the expression mechanisms in the p21X mRNA transcript and those in the other viral transcripts.

Base Sequence↗

PubMed1982

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts.

When resting (G0) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.

Animals↗

PubMed1988

Regulation of LH subunit mRNA levels by gonadal hormones in female rats.

To determine the physiological role of the ovaries in regulation of LH subunit gene expression, levels of cytoplasmic mRNA were measured in a cDNA-RNA dot-blot hybridization assay. An increase (twofold) in alpha mRNA was first detected 8 days after ovariectomy and then remained stable for 4 weeks. In contrast, LH-beta mRNA increased by 60-79% within 12 h of removing the ovaries and then rose progressively to six times the intact values at 3 and 4 weeks. Increases in LH-beta mRNA were always greater than those of alpha mRNA. Oestradiol, and oestradiol plus progesterone, but not progesterone alone, prevented the rise in alpha and LH-beta mRNA 10 days after ovariectomy. Three days after ovariectomy, alpha mRNA, but not LH-beta mRNA, was suppressed to below intact control values by oestradiol and oestradiol plus progesterone, indicating greater sensitivity of alpha mRNA to oestradiol inhibition at this stage. A single injection of oestradiol (1 microgram s.c.) to rats ovariectomized 14 days previously transiently suppressed alpha and LH-beta mRNA levels and serum LH concentrations in parallel for 1-8 h, after which high preinjection values were restored. However, pituitary LH content remained suppressed after LH mRNA levels had returned to the control values of ovariectomized rats. In most instances there was a qualitative positive correlation between changes in alpha and LH-beta mRNA, pituitary LH content and serum LH concentrations. LH content reflected LH-beta mRNA changes more closely than those of alpha mRNA. However, in oestradiol-treated rats ovariectomized 10 days previously, LH content remained increased despite normalization of the LH-beta and alpha mRNA levels, suggesting differential sensitivity to oestradiol of the gene expression and translational processes. Thus divergence of pre- and post-translational regulation of LH biosynthesis was demonstrated. These results imply an important physiological role for female sex hormones in the control of LH gene expression and LH biosynthesis. Prolactin mRNA fell by 30-50% for the first 2 weeks after ovariectomy, but by 3 and 4 weeks values were similar to those of intact controls. Serum and pituitary prolactin levels were reduced by 50% or more at all time-points, despite normalization of mRNA. Treatment of ovariectomized rats for 10 days with oestradiol and progesterone, either alone or combined, reversed the fall in prolactin mRNA and serum and pituitary prolactin levels. These changes in prolactin gene expression and synthesis were opposite to those of LH subunits in response to the same in-vivo hormone manipulations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2000

Expression and regulation of histidine decarboxylase mRNA expression in the uterus during pregnancy in the mouse.

It has been hypothesized that hormonally regulated histamine production plays a role in preparation of the uterus for implantation. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production. The current study was designed to determine intrauterine expression of HDC mRNA expression during pregnancy in the mouse. High levels of HDC mRNA expression were observed in the preimplantation mouse uterus with peak expression occurring on day 4. High levels of HDC mRNA expression were also detected in the post-implantation uterus. In an effort to determine whether HDC mRNA is regulated by pro-inflammatory cytokines, the HDC mRNA pattern was compared to intrauterine expression of mRNA's for interleukin-1alpha (IL-1alpha), IL-1beta, macrophage chemotactic protein-1 (MCP-1) and RANTES (regulated on activation, normal T expressed and secreted) during the peri-implantation period. IL-1beta, MCP-1 and RANTES mRNA levels were increased in the uterus on days 1-2 and on days 4-5. Increased expression of IL-1alpha mRNA was observed on days 1-2 and days 5-7. There was no clear relationship between HDC mRNA expression and cytokine/chemokine mRNA expression. Progesterone-stimulated intrauterine expression of HDC mRNA. Intrauterine cytokine/chemokine mRNA was also hormonally regulated. This data allowed the possibility that one or more of these pro-inflammatory cytokines could be involved in regulating intrauterine HDC mRNA production. Recombinant IL-1alpha, IL-1beta, MCP-1 and RANTES all failed to induce HDC mRNA expression in the preimplantation uterus in a mouse pseudopregnancy model. At the same time, IL-1beta induced the expression of mRNA for each of the four cytokines/chemokines. Despite the fact that these were also produced in the uterus during pregnancy and were hormonally regulated, none of these cytokines induced intrauterine HDC mRNA expression. The data suggest that progesterone is involved in the regulation of HDC mRNA expression in the preimplantation uterus, but IL-1alpha/beta, MCP-1 and RANTES, which have been reported to regulate histamine synthesis during inflammatory processes, do not appear to play a role.

Animals↗

PubMed1995

Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA.

The existence of opioid receptors within glial cell membranes has been proposed by several laboratories based on biochemical and radioligand binding data. The recent cloning of the mu, delta and kappa receptors has enabled us to directly examine the issue of opioid receptor expression in rat brain astroglia by using solution hybridization/ribonuclease protection assays to analyze the total RNA obtained from primary cultures of cortical, striatal, cerebellar, hippocampal and hypothalamic astrocytes. The results indicate that all five glial cultures expressed mu, delta and kappa receptor mRNA. The rank order of receptor mRNA abundance, expressed collectively across all five cultures, was determined to be delta > or = kappa >> mu. An analysis of the glial distribution profile for each receptor type revealed that mu receptor mRNA levels were the most abundantly expressed in cortical cultures, while the greatest levels of delta receptor mRNA were found in the cortical and hypothalamic cultures, and significant kappa receptor mRNA levels were produced by the cortical, hypothalamic and cerebellar cultures. Furthermore, the five glial cultures each expressed different levels of total opioid receptor (mu + delta + kappa) mRNA. The rank order of total opioid receptor mRNA expression across different astroglial cultures was found to be cortex > hypothalamus > cerebellum = hippocampus > striatum. An analysis of the relative expression profiles for mu, delta and kappa receptor mRNA within each culture revealed that all cultures manifested relatively high levels of delta and kappa receptor mRNA, but relatively low levels of mu receptor mRNA. Generally, cortical, hippocampal and hypothalamic cultures were characterized by comparable levels of delta and kappa receptor mRNA, and little, if any, mu receptor mRNA. However, striatal cultures were characterized by a high level of delta receptor mRNA which was approximately twice and four times that of the kappa and mu receptor mRNA, respectively. In contrast, cerebellar cultures expressed predominantly kappa receptor mRNA at a level which was almost twice that of the delta receptor mRNA, and expressed very little mu receptor mRNA. These data show that primary astroglial cultures not only express mu, delta and kappa receptor mRNAs, but they do so in a manner dependent upon receptor type and brain region. This suggests a regional heterogeneity of astrocytes with respect to opioid receptor expression, a characteristic previously described only for neurons. Furthermore, it suggests the existence of an additional anatomical component in CNS opioid systems.

Animals↗

PubMed2003

Detection of epidermal growth factor receptor mRNA in peripheral blood of cervical cancer patients.

OBJECTIVE: Epidermal growth factor receptor (EGFR) has been reported to be expressed by immunohistochemistry in invasive cervical cancers. We evaluated the feasibility of detecting EGFR mRNA by EGFR-based reverse transcription polymerase chain reaction (RT-PCR) in peripheral blood of patients with cervical cancer. METHODS: Expression of EGFR mRNA, cytokeratin (CK)-19 mRNA, and CK-20 mRNA was examined by RT-PCR in 12 human cervical cancer cell lines. All 12 cell lines expressed both EGFR mRNA and CK-19 mRNA, but only 4 of 12 (33.3%) cell lines expressed CK-20 mRNA. Peripheral blood samples from 20 healthy donors and 45 cervical cancer patients were also examined. RESULTS: In peripheral blood from 20 healthy donors, neither EGFR mRNA nor CK-20 mRNA was expressed, but CK-19 mRNA was expressed in 13 of 20 (65%). In contrast, EGFR mRNA was expressed in 12 of 45 (26.7%) patients with cervical cancer (P = 0.0071, 2 test, patient vs control). On the other hand, expression of EGFR was observed in 98% of tumor tissues by immunohistochemistry. CK-19 mRNA and CK-20 mRNA were found in 35 of 45 (77.8%) and 0 of 45 (0%) patients, respectively (NS, chi(2) test, patient vs control). The rate of detection of EGFR mRNA in peripheral blood correlated with FIGO stage (P = 0.049). CONCLUSION: Both CK-19 mRNA and CK-20 mRNA showed no diagnostic value as markers of circulating tumor cells in cervical cancers. However, EGFR mRNA in blood might be a useful marker of circulating tumor cells in cervical cancers.

Carcinoma, Squamous Cell↗

PubMed1998

Relationships of PPARgamma and PPARgamma2 mRNA levels to obesity, diabetes and hyperinsulinaemia in rhesus monkeys.

OBJECTIVE: To examine the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) together with CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL) and glucose transporter (GLUT4) mRNA in adipose tissue of rhesus monkeys in relation to obesity. DESIGN: Cloning of the PPARgamma1 and gamma2 cDNAs and analysis of PPARgamma, C/EBPalpha, LPL and GLUT4 mRNA levels in the adipose tissue of lean and obese monkeys. SUBJECTS: 28 rhesus monkeys (Macaca mulatta) with a wide range of body weights (9.2-22.6 kg) and with or without type 2 diabetes. MEASUREMENTS: Sequence of PPARgamma1 and gamma2. Tissue distribution of PPARgamma1 and gamma2. The mRNA levels of PPARgamma, C/EBPalpha, LPL and GLUT4 in adipose tissue. The ratio of PPARgamma2 mRNA to total PPARgamma mRNA. RESULTS: The monkey PPARgamma2 protein showed 99% identity with the human protein. PPARgamma1 mRNA was shown to be expressed in various tissues and most abundantly in adipose tissue. PPARgamma2 existed mainly in adipose tissue. A significant correlation between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and obesity was observed, whereas total PPARgamma mRNA levels showed no significant relationships to obesity. There was also a significant relationship between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and fasting plasma insulin concentration. The mRNA levels of C/EBPalpha, LPL and GLUT4 were highly correlated to that of total PPARgamma mRNA. They were also significantly correlated to the mRNA levels of PPARgamma1 and PPARgamma2. CONCLUSIONS: The ratio of PPARgamma2 mRNA to total PPARgamma mRNA is related to obesity in the rhesus monkey and mRNA expression of PPARgamma1, PPARgamma2, C/EBPalpha, LPL and GLUT4 appear to be coordinated in vivo.

Adipose Tissue↗

PubMed2003

Angiotensin II upregulates the expression of vasopressin V2 mRNA in the inner medullary collecting duct of the rat.

Previous in vivo studies in cardiomyopathic hamsters suggested that the expression of vasopressin (AVP) V2 mRNA is up- regulated by angiotensin II. The present study was performed to determine whether angiotensin II plays a role in regulating the expression of AVP V2 mRNA and aquaporin-2 (AQP2) mRNA in the inner medullary collecting duct (IMCD) of the male Wistar rat. The expression of AVP V2 mRNA and AQP2 mRNA in the IMCD was measured by competitive reverse-transcriptase polymerase chain reaction (RT-PCR). Six groups of experiments were performed. In the first group, we incubated IMCD with 3 different doses of angiotensin II (10(-11), 10(-9) and 10(-7) mol/L). Angiotensin II caused a significant increase in the AVP V2 mRNA in a dose-dependent manner but its effect on AQP2 mRNA was modest. This effect of angiotensin II was inhibited by angiotensin II receptor antagonist, [Sar1,Ile8]-angiotensin II. To examine the role of PKA in mediating an increase in AVP V2 mRNA expression, we incubated IMCD with 10(-7) and 10(-11) M of angiotensin II in the presence of a specific protein kinase A (PKA) inhibitor, Rp diasteroisomer of adenosine 3'-5'-cylic monophosphothionate (Rp-cAMPS). The angiotensin II-induced upregulation of V2 mRNA was abolished. In the fourth group, we examined the effect of protein kinase C (PKC) inhibition on V2 mRNA expression. The upregulation of V2 mRNA induced by angiotensin II was greatly exaggerated when IMCD was incubated with angiotensin II and RO-31-8220 (PKC inhibitor). In the fifth and sixth groups of studies, we determined the direct effect of PKA and PKC on regulating the expression of V2 mRNA and AQP2 mRNA in the IMCD, respectively. Dibutryl cAMP stimulated an upregulation in the expression of V2 mRNA and AQP2 mRNA, whereas phorbol esters suppressed the expression of V2 mRNA. These results suggested that PKA stimulates and PKC suppresses the expression of V2 mRNA in the IMCD of the kidney.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1996

Neural regulation of corticotropin releasing hormone (CRH) and CRH receptor mRNA in the hypothalamic paraventricular nucleus in the rat.

The role of afferent innervation to the hypothalamic paraventricular nucleus (PVN) on CRH mRNA and CRH receptor mRNA levels was studied in control and stressed rats. Groups of rats were subjected to unilateral transection of the stria terminalis (ST), the medial forebrain bundle at the rostral hypothalamic level (RMFB), or the lower brainstem through the medulla oblongata between the obex and the locus coeruleus (CBs). Twelve days after surgery, each group of rats was further divided into controls (basal conditions) and stressed (1 h immobilization), before collecting brains for mRNA analysis by in situ hybridization histochemistry. While ST and RMFB cuts had no effect on basal CRH mRNA levels in the PVN, CBs cut decreased CRH mRNA in the PVN ipsilaterally to the knife cut but it was without effect on the contralateral side (-40% and -37% vs contralateral and sham-operated, respectively, P < 0.01). Acute stress (rats were killed 3 h after immobilization) increased CRH mRNA levels by about 30% bilaterally, an effect which was unchanged by any of the three hemisections. Under basal conditions, CRH receptor mRNA levels in the PVN were indistinguishable from the surrounding areas in sham-operated controls, ST and RMFB operated rats. However, brainstem hemisection resulted in clear expression of CRH receptor mRNA in areas consistent with the dorsal, medial-ventral and lateral parvicellular subdivisions of the PVN, ipsilateral to the transection. CRH neurons in these subdivisions project to the lower brainstem and the spinal cord. Expression of CRH receptor mRNA in the medial-dorsal and anterior parvicellular divisions (CRH neurons with median eminence projections) was not affected by CBs cut. In these subdivisions, immobilization stress markedly increased CRH receptor mRNA levels but it did not influence CBs cut-induced CRH receptor expression. ST and RMFB hemisections were without effect on PVN CRH receptor mRNA levels under basal or stress conditions. Oxytocin (OT) and vasopressin (VP) mRNA levels in the magnocellular subdivision of the PVN were unchanged after immobilization, or following ST, RMFB or CBs cuts, whereas OT mRNA in the medial-ventral and caudal parvicellular subdivisions was decreased by 52% after CBs cut. The data demonstrate that: 1) basal CRH mRNA levels in the PVN are under tonic stimulatory influence of the lower brainstem (and/or spinal cord) afferents; 2) CRH receptor mRNA expression in PVN subdivisions (pituitary vs lower brainstem/spinal cord projecting neurons) is under different control mechanisms, and 3) immobilization-induced changes in CRH mRNA and CRH receptor mRNA levels are mediated either by neural inputs from brain areas other than those investigated here, or by humoral factors.

Animals↗

PubMed1983

Translational competition by mRNA species encoding albumin, ferritin, haemopexin and globin.

Messenger RNA from rat liver was translated in a micrococcal-nuclease-treated reticulocyte lysate supplemented with liver tRNA. Synthesis of the liver proteins haemopexin, ferritin and albumin was analyzed by quantitative immunoprecipitation. The relative translation yield of these proteins changed as a function of the amount of mRNA present during protein synthesis, revealing the existence of translational competition between individual species of mRNA from the liver. The results show that the mRNA species encoding haemopexin, ferritin and albumin possess distinctly different abilities to compete for one or more critical components in translation, with competitive strength increasing in this order. Although on a weight basis total liver mRNA is apparently as effective a template for protein synthesis as is globin mRNA, the latter displays a greater resistance to inhibition of its translation by KCl. In analogy with the translation properties of alpha-globin and beta-globin mRNA [Di Segni, G., Rosen, H. and Kaempfer, R. (1979) Biochemistry, 18, 2847-2854], this finding suggests that globin mRNA possesses greater competitive strength than does total liver mRNA. Increasing amounts of globin mRNA competitively inhibit the translation of albumin and ferritin mRNA present in total liver mRNA. The competition is relieved by the addition of eukaryotic initiation factor eIF-2. Translation of ferritin mRNA responds more vigorously to relief by eIF-2 than does translation of albumin mRNA, a finding consistent with the observation that albumin mRNA competes more effectively than ferritin mRNA in translation. The results support the assumption that albumin mRNA possesses a greater affinity for eIF-2 than does ferritin mRNA.

Albumins↗

PubMed1988

Regulation of translation and stability of an mRNA coding for a 40-kDa polypeptide in rat L6 muscle cells.

The mRNA coding for a 40-kDa polypeptide (P-40) was previously cloned and sub-cellular distribution of this mRNA was examined in rat L6 myoblast cells under different conditions [Pramanik, S. & Bag, J. (1987) Eur. J. Biochem. 170, 59-67]. The translation of this mRNA was found to be regulated during differentiation of myoblasts. This mRNA was translated in proliferating myoblasts but not in the non-dividing differentiated myotubes. We have further examined whether the mRNA present in the polysomal fraction of myoblasts and that in the free non-polysomal fraction in myotubes was identical by nuclease S1 mapping. The coding strand of the 600-base-pair PstI fragment of the recombinant clone was 3'-end-labeled with cordycepin 5'-[alpha-32P]triphosphate and hybridized with RNA from either myoblasts or myotubes. The results of these studies have shown that RNA from both preparations was fully able to hybridize with the probe DNA and, therefore, protected the 600-nucleotide-long fragment from nuclease S1 digestion, thus suggesting that the sequence of 600 nucleotides at 3' ends of both translationally active polysomal mRNA of myoblasts and repressed free mRNA of myotubes are identical. These results also confirmed the results of our earlier studies on the subcellular distribution of this mRNA by Northern blot analysis. Further studies were also performed to determine whether withdrawal of muscle cells from the cell cycle during differentiation to form myotubes alone was responsible for regulating translation of P-40 mRNA. The results of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by cytosine arabinoside have shown that translation of P-40 mRNA continued in absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P-40 mRNA in myotubes. The relationship between the translation of P-40 mRNA and its stability was examined. Two different methods were used to determine the stability of mRNAs. The first approach was by determining the steady-state levels of this mRNA following inhibition of RNA synthesis by actinomycin D. In the second method, we have determined the amount of 3H-labeled P-40 mRNA during pulse and chase experiments. Both methods produced similar results. It was found that the stability of P-40 mRNA was not altered during differentiation of rat L6 cells. The results of pulse and chase studies have also shown that P-40 mRNA was synthesized in both myoblasts and myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amanitins↗

PubMed1994

Effect of peripheral axotomy on expression of neuropeptide Y receptor mRNA in rat lumbar dorsal root ganglia.

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA-positive, and the majority of these neurons were of the small type, with only a few labelled medium-sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene-related peptide and galanin mRNAs in small neurons, but not in medium-sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA-positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA-positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA-positive large neuron profiles in the ipsilateral DRGs, and they constituted 15-20% of counted DRG neuron profiles and 45-65% of counted large neuron profiles, 7-28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium-sized neuron profiles, with galanin mRNA in some small, medium-sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium-sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA-positive small neurons. In the dorsal horn, NPY receptor mRNA-positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA-positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗