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At least 217 records · Page 12Linked to original sources
PubMed2003

Utility of the dried blood on filter paper as a source of cytokine mRNA for the analysis of immunoreactions in Plasmodium yoelii infection.

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

Animals↗
PubMed1975

Effects of withdrawal of a serum stimulus on the protein-synthesizing machinery of cultured fibroblasts.

3T6 cells resting in medium containing 0.5% serum were stimulated to prepare for multiplication by the addition of medium containing 10% serum. After a number of hours, when the rate of preribosomal RNA synthesis, total RNA content (mainly ribosomal), and the cytoplasmic content of poly A (a measure of poly A(+) mRNA) were considerably elevated, the serum-rich medium was withdrawn, and the original medium replaced. The rate of preribosomal RNA synthesis began to drop within 30 minutes, but required a much longer time to fall to a new resting level. When the serum-rich medium was withdrawn after 12 hours of stimulation, the total RNA content required 12-18 hours to fall to the resting level, whereas cytoplasmic poly A content and the rate of protein synthesis declined more rapidly, reaching a new resting level within eight hours. During the 12 hours following withdrawal of the serum-rich medium an appreciable fraction of the cells initiated DNA synthesis. Presumably, the cellular preparations for DNA synthesis cannot be immediately reversed because of the inertial factors related to the protein-synthesizing machinery.

Blood↗
PubMed1990

Amplification of the c-myc and the pvt-like region in human multiple myeloma.

Genetic alterations that lead to the clonal expansion of differentiated cells in multiple myeloma have still to be elucidated. Many chromosomal aberrations have been found, but until now, none of them is typically associated with multiple myeloma. In search for genetic defects in multiple myeloma we studied the structure and expression of the c-myc oncogene and the pvt-like region because of their frequent association with other B-cell malignancies. Here we report co-amplification of the c-myc oncogene and the 5' part of the pvt-like region in two out of 26 cases of multiple myeloma. In both cases only kappa-light chains were produced. The amplification also manifested itself at the RNA level. Total RNA was analysed in one of these two cases showing abundant c-myc mRNA. In the same RNA sample we also detected a strong hybridizing band of about 7 kb, when the pSS.4 probe, representing the 5' part of the pvt-like region, was used. This band was not present in total RNA from normal bone marrow cells or bone marrow from multiple myeloma patients without the amplification of c-myc and the pvt-like region. Until now, transcripts of the pvt-like region were only found in a few human cell lines ranging in size from 1 to 11 kb. This is the first case in which a high expression of a +/- 7 kb transcript of the pvt-like region has been found in freshly obtained tumor material probably due to a pvt-amplification. The occurrence of abnormalities in the c-myc and the pvt-like region in multiple myeloma is a rare event and may be associated with the progression of this type of tumor.

DNA, Neoplasm↗
PubMedDate not supplied

Separation technique for messenger RNAs by use of schizophyllan/poly(A) tail complexation.

Schizophyllan (SPG) is one of the water soluble beta-1,3-glucans and has a peculiar molecular recognition capability, namely, the single stranded SPG (s-SPG) can form a stoichiometric complex with certain polynucleotides such as poly(C) and poly(A), although it cannot bind poly(G) and poly(dC) at all. In this paper, we prepared an s-SPG-appended column and made an attempt to separate polynucleotides on the bases of this molecular recognition capability. The s-SPG-appended column trapped only such RNAs that could form the complex with s-SPG but eluted other RNAs which did not form the complex. Encouraged by the results in the model system, we extended the s-SPG-appended column into separation of native messenger RNAs (mRNAs) from a RNA mixture (total RNA) obtained from yeast. Since eukaryotic mRNAs have a poly(A) tail with 150-300 bases, we supposed that the tails would be trapped by the s-SPG-appended column. The results indicate that mRNAs were separated from total RNA in good yield and with high purity. It should be emphasized that this is the first device to separate natural mRNAs without using a dA/dT Watson-Crick-type interaction.

Carbohydrate Sequence↗
PubMed1983

Guanosine 5'-diphosphate 3'-diphosphate levels, carbon source, and ribonucleic acid synthesis in a mutant strain of Escherichia coli.

We have previously described a mutant strain of Escherichia coli (2S142) which shows a specific inhibition of stable RNA synthesis at 42 degrees C. The temperature-sensitive lesion mimics a carbon source downshift (diauxie lag). We therefore measured RNA synthesis and levels of ppGpp (guanosine 5'-diphosphate 3'-diphosphate) on a number of different carbon sources. There is a 6-fold variation in ppGpp levels at 42 degrees C, depending on the carbon source present. Much of the variation in ppGpp levels at 42 degrees C can be explained by variations in the decay rate of ppGpp at 42 degrees C. The rates of ribosomal RNA and total RNA synthesis also vary with the carbon source at 42 degrees C. Linear regression analysis shows only a moderately good correlation (correlation coefficient = 0.62, P = 0.0001) between the ppGpp level at 42 degrees C and the rate of rRNA synthesis at 42 degrees C. In fact, ppGpp levels are a slightly better predictor of the rate of total RNA synthesis (correlation coefficient = 0.69, P = 0.0001) at 42 degrees C. Other variables such as rate of carbon source uptake appear to have very little, if any, relationship to the rate of rRNA synthesis on the different carbon sources. Segmented linear regression analysis indicates that ppGpp levels and rates of RNA synthesis correlate best when the carbon sources are divided into two groups: 6- and 12-carbon sugars and other carbon sources. The rate of rRNA synthesis in 2S142 at 42 degrees C appears to be relatively insensitive to ppGpp levels with 6- and 12-carbon sugars as the carbon source. These data raise the possibility that carbon source may affect rRNA synthesis in a manner that is at least partially unrelated to ppGpp levels.

Carbohydrate Metabolism↗
PubMed2004

Microarray gene analysis of the liver in a rat model of chronic, voluntary alcohol intake.

The mechanisms underlying alcoholic liver disease are not fully understood. It has been established that alcohol interferes with transcriptional and translational regulatory steps of cell function. To understand such an effect, assessment of alcohol-induced changes in the simultaneous expression of a large number of genes may prove very useful. The purpose of the current study was to test a large number of genes ( approximately 8700) for possible changes in expression induced by alcohol alone or in addition to treatment with lipopolysaccharide (LPS), a putative mediator of alcohol effects on the liver. Male rats were fed an alcohol-containing liquid diet (Lieber-DeCarli) for 14-15 weeks, injected with Escherichia coli LPS (0.8 mg x kg(-1)), and killed 24 h later. Blood samples were taken for determination of plasma liver enzyme activity, and liver samples were obtained for histologic evaluation and total RNA extraction. Total RNA was analyzed for gene expression (Rat Toxicology U34 Array; Affymetrix, Santa Clara, CA). Of 8740 genes on the microchip, 2259 were expressed in the liver. Seven hundred ninety-eight genes underwent significant changes induced by either alcohol or LPS, but listed in this article are only those that significantly increased or decreased expression twofold or more. The genes were assigned to functional groups and reviewed. Gene changes were discussed from two viewpoints: relevance to established hypotheses of alcohol and LPS mechanisms of action and revealing of novel mechanisms of alcohol-induced liver injury. Application of DNA microarray technology to the study of alcohol-induced liver injury generated novel theoretical and experimental approaches to alcohol-induced liver injury.

Alcohol Drinking↗
PubMed2005

Gene expression profiling of HIV-1 infection using cDNA microarrays.

To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dye-specific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section.

CD4-Positive T-Lymphocytes↗
PubMed2004

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

Recent studies in our laboratory have demonstrated that bosentan, a mixed endothelin ET(A)/ET(B) receptor antagonist, prevented the upregulation of the arginine vasopressin (AVP) V(2) receptor in the inner medullary collecting duct (IMCD) of cardiomyopathic hamsters. These results suggested that endothelin-1 (ET-1) is involved in the upregulation of AVP V(2) receptors. Studies were performed to detect the effect of ET-1 on the expression of AVP V(2) receptors and the ET receptor mediating these effects within the IMCD of the rat. Rat IMCD tissue was isolated and incubated with the following: ET-1, or ET-1 in combination with ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788, respectively, and sarafotoxin c (S6c), an ET(B) receptor-specific agonist. Tissue samples were then analyzed using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. ET-1 treatment resulted in increased V(2) mRNA from a control level of 186.8 +/- 15.0 amol/microg total RNA to 430.7 +/- 49.0 amol/microg total RNA (P <.003). ET-1/ET(A) treatment resulted in no significant decrease in V(2) mRNA expression 335.0 +/- 38.0 amol/microg total RNA. Whereas ET-1/ET(B), and ET-1/ET(B)/ET(A) treatment resulted in V(2) mRNA approaching control 256.0 +/- 15.0 amol/microg total RNA, and 215.6 +/- 42.3 amol/microg total RNA. However, ET-3 treatment produced no significant changes in V(2) receptor mRNA expression. Sarafotoxin treatment corroborated both the ET-1 and ET receptor antagonist data, demonstrating striking significant increases in V(2) receptor mRNA and protein expression. S6c treatment increased V(2) mRNA expression from a control level of 199 +/- 17.3 amol/microg total RNA to 284.3 +/- 42.1 amol/microg total RNA (P < 05). Western blotting revealed that changes in V(2) mRNA expression in the various treatment conditions were similar to changes in protein expression. Overall, these data indicate that in the IMCD ET-1 increases AVP V(2) receptor expression and these changes are mediated by the ET(B) receptor.

Animals↗
PubMed1980

Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy.

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.

Acridine Orange↗
PubMed1985

Ascorbate-generated endogenous extracellular matrix affects cell protein synthesis in calf aortic smooth muscle cells.

Ascorbate supplementation of cultured fetal calf aortic smooth muscle cells leads to increased deposition of extracellular matrix proteins and stimulation of cellular protein synthesis (E. Schwartz et al., J cell biol 92 (1983) 462) [7]. In the present study, we have investigated this phenomenon at the level of gene expression. Cells were grown for three weeks on tissue culture plastic with or without ascorbate (50 micrograms/ml). When compared to controls, cells grown in presence of ascorbate had twice as much poly(A+) RNA per microgram of total RNA, and ascorbate led to a 50% increase in [35S]methionine incorporation when the total RNA was translated in the reticulocyte lysate system. SDS-PAGE revealed no change in the protein pattern under the two conditions. "Northern" hybridization revealed a two- to fivefold increase in the sequence content of beta-actin, alpha-tubulin and type I pro alpha 1-collagen in total RNA of ascorbate-supplemented cells, but no difference was observed in the mRNA sequence content for the three specific proteins when equal amounts of poly(A+) RNA from ascorbate and control cells were hybridized with the three cloned cDNAs. To evaluate the effect of an exogenous matrix, cells were also plated on collagen gels. RNA isolated from cells grown on collagen without added ascorbate exhibited translational activity and mRNA sequence content similar to cells grown with ascorbate on tissue culture plastic. In contrast, no differences from controls were found in cells grown for one week in the presence of ascorbate, at which time no significant deposition of collagen occurs in the extracellular matrix. These results suggest that the stimulation in protein synthesis in fetal calf smooth muscle cells supplemented with ascorbate is associated with an increase in the proportion of poly(A+) RNA in the total RNA pool, and that the production of an endogenous collagen-rich matrix in the presence of ascorbate may be the basis for these pretranslational changes.

Actins↗
PubMed2002

A highly reproducible, linear, and automated sample preparation method for DNA microarrays.

DNA microarrays are powerful tools to detect changes in transcript abundance in multiple samples in parallel. However, detection of differential transcript levels requires a reproducible sample (target) preparation method in addition to a high-performance microarray. Therefore, we optimized a target-preparation method that converts the poly(A)(+) RNA fraction of total RNA into complementary DNA, then generates biotin-labeled complementary RNA from the cDNA. We measured the efficiency of incorporation of biotin-containing nucleotides by an enzymatic digestion, followed by resolution via analytical high-performance liquid chromatography (HPLC). When the target was hybridized to a sensitive and reproducible microarray platform, low coefficients of variation in both hybridization intensities and differential expression ratios across target preparations were observed. Nearly identical hybridization intensities and expression ratios are observed regardless of whether poly(A)(+)-enriched RNA or total RNA is used as the starting material. We show the ability to discern biological and production variability through the use of different lots of commercial samples as visualized by hierarchical clustering. Automation of the target-preparation procedure shows equivalence to the manual procedure, reproducible yields of target, and low variability as measured by hybridization to microarrays. Most importantly, RNA mixing experiments show a linear and quantitative amplification in probe hybridization signals for >6000 genes across the entire signal range.

Biotinylation↗
PubMed1979

Isolation and Cell-free Translation of Total Messenger RNA from Germinating Castor Bean Endosperm.

Polyadenylated RNA was isolated from the total RNA fraction extracted from the endosperm tissue of 3-day-old castor bean seedlings by affinity chromatography on oligo(dT)-cellulose. This polyadenylated RNA was efficiently translated into protein when added to a messenger RNA-dependent cell-free system derived from rabbit reticulocytes. Characterization of the translational products by electrophoresis followed by autoradiography established that numerous discrete polypeptides were formed with molecular weights ranging from 10,000 to over 100,000. Immunoprecipitation in the presence of antiserum raised in rabbits against the total glyoxysomal matrix proteins showed that these proteins accounted for 15 to 20% of the total translational products.Attempts to reconstitute rough endoplasmic reticulum by the addition of washed castor bean microsomal membranes to the translational system were unsuccessful, these membranes severely inhibiting protein synthesis. Canine pancreatic microsomes could be added to endosperm messenger RNA-dependent reticulocyte lysates at relatively high concentrations while still allowing significant protein synthesis.

Journal Article↗
PubMed1990

Human thymic epithelial cells produce IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor.

The development of conditions for culturing normal human thymic epithelial (TE) cells free from contaminating stromal cells has allowed us to characterize a number of cytokines produced by TE cells. Using cDNA probes for human IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor (LIF), we identified mRNA for these cytokines by RNA blot analysis of total RNA preparations derived from TE cells. We demonstrated that TE cells produced IL-6 transcripts and that TE cell culture supernatants contained IL-6 biologic activity, as determined by the ability to support proliferation of the T1165 plasmacytoma line. The 1.0-kilobase (kb) transcript of granulocyte-monocyte-CSF was also detected in TE cell-derived total RNA. TE cell culture supernatants contained LIF activity, as determined by proliferation of the murine cell line DA-1a, and a 4.0-kb LIF transcript was detected in TE cell-derived total RNA preparations. The 4.0-kb LIF transcript from TE cell-derived total RNA corresponded in size to the LIF transcripts in PMA-activated T lymphocytes. Thus, using biologic assays and RNA blot analysis, we demonstrated that cultured normal human TE cells produced both immunoregulatory cytokines and cytokines that drive various differentiation stages of human hematopoiesis. Our findings support the hypothesis that TE cells may play a role in providing cytokines that are important for the proliferation and differentiation of hematopoietic precursor cells that migrate to the thymus during fetal and postnatal human thymic development.

Antigens, CD7↗
PubMed2005

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR.

BACKGROUND: Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target. RESULTS: We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65 degrees C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step. CONCLUSION: Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.

Actins↗
PubMed1997

Gonadectomy alters tyrosine hydroxylase and norepinephrine transporter mRNA levels in the locus coeruleus in rabbits.

The central noradrenergic system has a major regulatory role on gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion in rabbits. Exogenous administration of norepinephrine (NE) alters GnRH/LH release in a sex steroid-dependent manner, i.e. NE stimulates GnRH/LH release in oestrogen-primed ovariectomized (OVX) animals but not in non-primed individuals. To investigate how gonadal steroids influence noradrenergic neuronal activities in the locus coeruleus (LC), mRNA levels of tyrosine hydroxylase (TH) and NE transporter (NET), two key factors regulating NE synthesis and uptake, were compared 3 weeks after gonadectomy (GDX). Intact male (n = 5) and female (n = 6) New Zealand White rabbits were sacrificed along with castrated males (n = 4) and OVX females (n = 5). The brainstem from each individual was sectioned and the LC was punched for detection of TH and NET mRNA levels using the ribonuclease protection assay (RPA). Trunk blood was collected to determine immunoactive serum LH values. Levels of LH were elevated in both males and females after GDX. Luteinizing hormone concentrations averaged 0.10 +/- 0.05 ng/ml in intact males vs 1.64 +/- 0.31 ng/ml in castrated males (P < 0.01) and 0.30 +/- 0.08 ng/ml in intact vs 9.80 +/- 3.50 ng/ml in OVX females (P < 0.05), respectively. Removal of the gonads also increased TH mRNA levels in the LC in both males and females. In intact males, TH mRNA levels were 0.796 +/- 0.181 pg/microgram total RNA, whereas in castrates mRNA levels averaged 1.667 +/- 0.345 pg/microgram total RNA (P < 0.05). In intact females, TH mRNA levels were 0.617 +/- 0.054 pg/microgram total RNA while the OVX group averaged 1.084 +/- 0.202 pg/microgram total RNA (P < 0.05). Similar increases in NET mRNA were noted after GDX in both sexes. In males, NET mRNA levels were 1.461 +/- 0.401 pg/microgram total RNA in intacts vs 3.666 +/- 0.649 pg/microgram total RNA in castrates (P < 0.05). In females, NET mRNA levels averaged 1.336 +/- 0.212 pg/microgram total RNA and 3.297 +/- 0.835 pg/microgram total RNA in the intact and OVX groups, respectively (P < 0.05). The data indicate that GDX enhances gene expression of both TH and NET. The results support the hypothesis that the feedback regulation of sex steroids on LH secretion in rabbits of both sexes involves transcriptional/translational processes of at least TH and NET in brainstem NE cells.

Animals↗
PubMedDate not supplied

[Secondary structure of total protein and 23S RNA in 50S ribosomal subunits and in the isolated state].

Optical and sedimentational studies of isolated 23S RNA, total proteins and some RNP-complexes of the 50S subunits were carried out. It is shown that the secondary structure content of 23S RNA in the ribosome is lower than in the isolated state. Ribosomal proteins stabilize the 23S RNA structure and make it more compact. At the same time they cause some unwinding effect on the secondary structure of the 23S RNA and possibly fix some segments of the 23S RNA in the conformation necessary for its function. In turn, the 23S RNA increased somewhat the level of the total ordered secondary structure in the ribosomal proteins. There was no considerable change of the ratio between the alpha- and beta-structures in the proteins.

Bacterial Proteins↗
PubMed2006

[Involvement of matrix metalloproteinase-2, -9, and tissue inhibitors of metalloproteinase-1, 2 in occurrence of the accrete placenta].

OBJECTIVE: To investigate the roles of matrix metalloproteinase-9, -2 (MMP-9, 2), and tissue inhibitors of metalloproteinase-1, 2 (TIMP-1, 2) in pathogenesis of the accretio placenta. METHODS: The women with the placenta accrete were recruited and the placenta (23) and deciduas tissues (9) after labor were obtained, and the placenta (28) and deciduas (11) from women without the placenta accreta were obtained as control to get, too. The expressions of MMP-9, -2, TIMP-1, 2 in the placental and decidual tissues were analyzed by real-time PCR. RESULTS: mRNA expression of MMP-9 in the placenta accreta was (3.21 +/- 0.76) copies/microg total RNA, significantly higher (P < 0.05) than that of normal placenta [(3.84 +/- 0.24) copies/microg total RNA)]. MMP-9 transcription in the decidua accreta was (2.50 +/- 0.49) copies/microg total RNA, significantly higher (P < 0.05) than that of normal decidua [(3.81 +/- 0.66) copies/microg total RNA]. mRNA expression of TIMP-1 in normal placenta and placenta accreta was (5.91 +/- 0.56) and (5.92 +/- 0.46) copies/microg total RNA, respectively, with no significant difference between the two groups. mRNA expression of TIMP-1 in the accrete deciduas was (6.63 +/- 0.51) copies/microg total RNA, significantly lower (P < 0.05) than that of normal decidua (7.09 +/- 0.55) copies/microg. mRNA expression of MMP-2 in the accrete placenta was (4.55 +/- 1.13) copies/microg total RNA, significantly higher (P < 0.05) than that of normal placenta (5.53 +/- 0.59) copies/microg. mRNA expression of MMP-2 in the accrete decidua and normal decidua was (6.07 +/- 0.83) and (5.97 +/- 0.76) copies/microg total RNA, respectively, with no significant difference between the two groups. mRNA expression of TIMP-2 in the accrete placenta was (4.69 +/- 0.60) copies/microg total RNA, significantly higher (P < 0.05) than that of normal placenta (3.79 +/- 1.06) copies/microg. mRNA expression of TIMP-2 in the accrete decidua was (5.06 +/- 0.33) copies/microg total RNA, higher significantly (P < 0.05) than that of normal decidua (3.98 +/- 0.60) copies/microg. CONCLUSIONS: The upregulation of MMP-9, MMP-2 in placenta and downregulation of TIMP-1 in decidua were involved in occurrence of the placental accreta, and the roles of TIMP-2 in occurrence of the placental accreta need to elucidated.

Adult↗
PubMed1999

A PCR analysis of ERalpha and ERbeta mRNA abundance in rats and the effect of ovariectomy.

To study the relative abundance and the changes of both estrogen receptor alpha (ERalpha) and ERbeta mRNA before and after ovariectomy in major organs important to the regulation of calcium homeostasis, we compared the degree of mRNA expression of ERalpha to that of ERbeta in rat tissues by performing competitive reverse transcription polymerase chain reaction (RT-PCR) with internal standards. Both ERalpha and ERbeta were highly expressed in the ovary {ERalpha[(2.2 +/- 0.33) x 10(7) copies/microg of total RNA] > ERbeta[(1.2 +/- 0.33) x 10(5) copies/microg of total RNA]} as we expected. The bone marrow and renal cortex were very important target organs of estrogen because ERalpha was highly expressed approximately 2 x 10(5) copies/microg of total RNA, but marrow cells revealed only a very weak expression of ERbeta [(0.7 +/- 0.21) x 10(2) copies/microg of total RNA]. Both ERalpha and ERbeta were expressed in the trabecular bone [(3.2 +/- 0.56) x 10(3) copy/microg of RNA] and [(2.8 +/- 0.21) x 102 copy/microg of RNA], respectively. However, they were not detected in the cortical bone. In the jejunum, the expression of ERalpha was not detectable, while ERbeta was expressed very weakly [(1.1 +/- 0.24) x 10(2) copies/microg of total RNA]. The thyroid gland expressed low copy numbers of ERbeta [(6.0 +/- 0.23) x 10(2) copies/microg of total RNA], but the parathyroid gland was negative for both ERalpha and ERbeta mRNA. In cultured stromal cells, ERalpha and ERbeta mRNAs were not detected after a 24-h culture; however, the rates of mRNA expression of ERalpha and ERbeta reached approximately 105 copies/microg of total RNA and approximately 10(2) copies/microg of total RNA, respectively, after 9-, 11-, and 13-day cultures. After ovariectomy, the expression of ERalpha mRNA decreased abruptly in the bone marrow and renal cortex, and both ERalpha and ERbeta were barely detected in the trabecular bone. In conclusion, ERalpha might be the main ER in organs important for calcium homeostasis, except in the jejunum. The mRNA expression of ERalpha in the bone marrow and renal cortex decreased abruptly after ovariectomy, which may partially explain why the effect of estrogen deficiency can be amplified and why trabecular bone loss is more predominant than cortical bone loss shortly after surgical or natural menopause.

Animals↗