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At least 433 records · Page 24Linked to original sources
PubMed2000

Regulation of corticotropin-releasing factor receptor type 2 beta messenger ribonucleic acid in the rat cardiovascular system by urocortin, glucocorticoids, and cytokines.

CRF receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart is modulated by exposure to both the bacterial endotoxin lipopolysaccharide (LPS) and glucocorticoids. In this study we examined the roles of glucocorticoids, cytokines, and CRF R2beta ligands in the regulation of CRF R2beta expression in the cardiovascular system both in vivo and in vitro. Using ribonuclease protection assays, we found that, in addition to the injection of LPS or corticosterone, physical restraint caused a decrease in CRF R2beta mRNA levels in the rat heart and aorta. Adrenalectomy with corticosterone replacement at constant levels partially blocked LPS-induced decreases in CRF R2beta mRNA expression in the heart. Thus, elevations of endogenous circulating corticosterone could contribute to the down-regulation of CRF R2beta mRNA expression in heart. To identify other putative modulating factors, we examined CRF R2beta expression in the aorta-derived A7R5 cell line. Incubation with CRF R2 ligands or dexamethasone reduced CRF R2beta mRNA levels. In addition, incubation with a variety of cytokines, proteins released during immune challenge, also reduced CRF R2beta mRNA expression. The multifactorial regulation of CRF R2beta mRNA expression in the cardiovascular system may serve to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.

Adrenocorticotropic Hormone↗
PubMed2000

Cloning and characterization of IL-17B and IL-17C, two new members of the IL-17 cytokine family.

IL-17 is a T cell-derived cytokine that may play an important role in the initiation or maintenance of the proinflammatory response. Whereas expression of IL-17 is restricted to activated T cells, the IL-17 receptor is found to be widely expressed, a finding consistent with the pleiotropic activities of IL-17. We have cloned and expressed two novel human cytokines, IL-17B and IL-17C, that are related to IL-17 ( approximately 27% amino acid identity). IL-17B mRNA is expressed in adult pancreas, small intestine, and stomach, whereas IL-17C mRNA is not detected by RNA blot hybridization of several adult tissues. No expression of IL-17B or IL-17C mRNA is found in activated T cells. In a survey of cytokine induction, IL-17B and IL-17C stimulate the release of tumor necrosis factor alpha and IL-1beta from the monocytic cell line, THP-1, whereas IL-17 has only a weak effect in this system. No induction of IL-1alpha, IL-6, IFN-gamma, or granulocyte colony-stimulating factor is found in THP-1 cells. Fluorescence-activated cell sorter analysis shows that IL-17B and IL-17C bind to THP-1 cells. Conversely, IL-17B and IL-17C are not active in an IL-17 assay or the stimulation of IL-6 release from human fibroblasts and do not bind to the human IL-17 receptor extracellular domain. These data show that there is a family of IL-17-related cytokines differing in patterns of expression and proinflammatory responses that may be transduced through a cognate set of cell surface receptors.

Amino Acid Sequence↗
PubMed2003

Regulation of inhibin alpha- and beta(A)-subunit messenger ribonucleic acid levels by gonadotropins and IGF-I in cultured chicken granulosa cells.

A quantitative competitive reverse transcription polymerase chain reaction assay (QC RT-PCR) for quantifying the absolute levels of the expression of inhibin alpha- and beta(A)-subunits in chicken granulosa cells showed that these subunits are expressed in different amounts depending on follicular maturation. The present study determined the regulation of the expression of these subunits. The individual effect of different doses of IGF-I, LH or FSH (1-100 ng/ml) or the combination of IGF-I with either LH or FSH at different concentrations, on the expression of inhibin alpha- and beta(A)-subunit was determined on cultured granulosa cells of F(1) and the combined F(4)+F(5) follicle. Cells were cultured for 48 h in 6-well plates with or without added hormones. Culture medium was discarded, cells were washed and total RNA was extracted from the cells. Five hundred nanograms of total RNA was reverse transcribed using specific primers and coamplified with an internal standard, as described previously, to determine expression level in the cells. IGF-I, LH, and FSH enhanced the inhibin alpha-subunit mRNA levels in a dose dependent manner in both F(1) and the combined F(4)+F(5) whereas inhibin beta(A)-subunit was not affected. The effects of FSH, LH were more expressed in F(1) follicles compared to F(4)+F(5) on the alpha-subunit. The addition of IGF-I and either LH or FSH during the culture period significantly increased the stimulatory effects of both LH and FSH on the expression of inhibin alpha-subunit in F(1) follicles but had no significant effect on the inhibin beta(A)-subunit. The results suggest that the changing expression levels of inhibin alpha-subunit during follicular development are the result of the regulatory effect of the interaction between IGF-I and the gonadotropins and that the regulation of this subunit may be the main factor for the regulation of the protein inhibin levels. Other factors may be also implicated in the changing expression levels of the beta(A)-subunit.

Animals↗
PubMed2004

5-HT7 receptor efficacy distribution throughout the canine stomach.

This study aimed to determine, quantify and explain regional differences in the relaxant response to the selective 5-HT(1) and 5-HT(7) receptor agonist 5-carboxamidotryptamine (5-CT) throughout the canine stomach. Longitudinal muscle strips from eight gastric corpus regions and six antrum regions were mounted for isotonic measurement. The 5-CT-induced relaxation was examined on a prostaglandin F(2alpha)-induced submaximal response, expressed as percentage of this response and fitted to the operational model of agonism (OMOA). 5-HT(7) receptor messenger RNA (mRNA) expression was compared by means of quantitative PCR. 5-CT inhibited PGF(2alpha)-induced tonic contraction (corpus) and increase of phasic contraction amplitude (antrum). The consistent antagonism produced by the selective 5-HT(7) receptor antagonist SB-269970 (10 nm, pA(2) estimates 8.2-8.9) confirmed that in every region, the inhibition by 5-CT was 5-HT(7) receptor mediated. However, variation in the maximum effect (61-108%) and pEC(50) (6.4-8.6) was observed throughout the different regions. The OMOA explained these differences as differences in the efficacy parameter tau (ratio of receptor density and coupling efficiency; log tau estimates ranging from 0.1 to 2.1). The log tau gradient decreases going from the lesser to the greater curvature. A proportional difference (68%) in the relative expression of 5-HT(7) receptor mRNA between the lesser and the greater curvature indicates that differences in receptor density contribute to the observed functional differences. This study illustrates that 5-HT(7) receptors are present throughout the ventral wall of the canine stomach, but the efficacy (expressed as log tau) is clearly greater close to the lesser curvature. Differences in 5-HT(7) receptor expression at least partially explain the functional differences.

Alternative Splicing↗
PubMed1998

Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT1 receptor subtypes to adenylyl cyclase.

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10-12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 microM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

Adenylyl Cyclases↗
PubMed2006

The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells.

Upregulation of functional voltage-gated Na+ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the "neonatal" splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by approximately 90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (approximately 30%), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33%, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43%, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42%, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.

Blotting, Western↗
PubMed2000

Expression of the leptin receptor during germ cell development in the mouse testis.

Leptin, a recently identified hormonal product of the ob gene, is known to regulate appetite, body metabolism, and reproductive functions. We investigated the expression of the leptin receptor (Ob-R) in testes from different age groups. The messenger RNA for Ob-R was found in testes from all age groups using RT-PCR. Using immunohistochemistry, we observed age- and stage-dependent distribution of the Ob-R in mouse testis. In testis of 5-day-old mice, its expression was mainly in type A spermatogonia. In the 20- and 30-day-old testis, Ob-R expression was in the spermatocytes; in the adult testis, it was specific to spermatocytes in stages IX and X of the cycle of the seminiferous epithelium. Five main immunoreactive proteins were detected using Western blot (220, 120, 90, 66, and 46 kDa). The 120-kDa protein was evident only in 20-day-old and older testes, whereas the 90-kDa band was present only in the 5- and 10-day-old testis. Leptin treatment induced phosphorylation of signal transducer and activator of transcription-3 in cultured seminiferous tubules from adult and 5-day-old testes. Our results show for the first time age- and stage-specific localization of a functional Ob-R in testicular germ cells. We hypothesize a direct role for leptin, through phosphorylation of signal transducer and activator of transcription-3, in proliferation and differentiation of germ cells, which may partially explain the infertility observed in leptin-deficient mice.

Aging↗
PubMed2001

Elevated mRNA expression for supervillin and vascular endothelial growth factor in human neutrophils stimulated with lipopolysaccharide from Porphyromonas gingivalis.

Differential gene expression was examined in human neutrophils stimulated with lipopolysaccharide from Porphyromonas gingivalis (P. gingivalis-LPS) using RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR). LPS from Escherichia coli (E. coli-LPS) was used as control. More than 200 differently expressed transcripts were found in 8 subjects showing differential regulation at the transcriptional level. Densitometric analyses revealed that 42-100 genes were upregulated, while 53-116 genes were downregulated by P. gingivalis-LPS compared with E. coli-LPS. The results of sequencing identification showed the presence of supervillin (SVIL) and vascular endothelial growth factor (VEGF) genes in the clones which were upregulated by P. gingivalis-LPS. Consequently, semiquantitative analyses proved that the level of mRNA for SVIL and VEGF were significantly higher in P. gingivalis-LPS-stimulated neutrophils than in other bacterial (E. coli, Actinobacillus actinomycetemcomitans, Prevotella intermedia) LPS- or synthetic lipid A-stimulated neutrophils. Our findings suggest that an elevated mRNA expression for SVIL could be associated with impaired function of neutrophils when stimulated by P. gingivalis-LPS. Further, the VEGF mRNA over-expression might be related to the pathogenesis of P. gingivalis-associated periodontitis. The RAP-PCR technique used in this study enabled us to identify a number of P. gingivalis-LPS regulated genes which hitherto have not been reported.

Adult↗
PubMed2003

Molecular cloning and characterization of rat chemokine-like factor 1 and 2.

Chemokine-like factor 1(CKLF1) is a newly cloned cytokine with three RNA splicing isoforms. It has chemotactic activities on leukocytes and plays an important role in skeletal muscle regeneration. Here we have isolated two rat homologues of human chemokine-like factors by expressed sequence tag assembly, which are designated as rat chemokine-like factor 1 and 2 (rat CKLF1, CKLF2). The full-length cDNAs of rat CKLF1 and -2 contain 523 and 682 nucleotides and the open reading frames encoding 98 and 151 amino acids, respectively. Rat CKLF1 and -2 share about 54.1 and 59.6% homologies with human CKLF1 and -2 at the amino acid level; both rat CKLF1 and -2 contain a CX3C motif at their C-terminal regions while human CKLFs have a CC motif at the same regions. Rat CKLFs are highly expressed in testis, while human CKLFs have a broad expression spectrum across multiple tissues. Recombinant rat CKLF1 can be secreted into the cell culture supernatants and has chemotactic effects on neutrophils, macrophages and lymphocytes, which is similar to human CKLF1, while recombinant rat CKLF2 has weaker chemotactic effects on these cells. These findings show that rat CKLFs have similar bioactivity with human CKLFs, although they are different in tissue distribution and contain different characteristic motifs.

Amino Acid Sequence↗
PubMed2001

Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

Amino Acid Sequence↗
PubMed2005

Ovarian carcinomas: CCN genes are aberrantly expressed and CCN1 promotes proliferation of these cells.

PURPOSE: The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family consists of six matricellular proteins that are involved in various cellular functions, such as proliferation, development, and angiogenesis. The purpose of this study was to explore the possibility that CCN genes are involved in ovarian cancers. EXPERIMENTAL DESIGN: We quantified CCN expression in a series of 59 ovarian cancers using quantitative real-time reverse transcription-PCR. CCN1 protein levels were further determined by immunohistochemistry and Western blot analysis. Overexpression and inhibition of CCN1 expression by small interfering RNA were used to examine its role in ovarian cancer cell proliferation in vitro and in vivo. RESULTS: We found dysregulation of levels of the various CCN mRNAs in ovarian cancers compared with their expression in normal whole ovaries. Expression of CCN1 protein was detected in normal ovarian epithelial cells and ovarian tumors as well as in ovarian cancer cell lines. Furthermore, estrogen increased CCN1 mRNA and protein levels in ovarian cancer cells. Ectopic expression of CCN1 enhanced the growth of ovarian cancer cells in liquid culture, whereas inhibition of its expression decreased proliferation and increased apoptosis in these cells. The observed changes in cell growth were accompanied with activation of Akt and extracellular signal-regulated kinase (ERK) signaling pathways. Stable expression of CCN1 in SKOV3 cells significantly increased tumorigenicity in nude mice. Finally, overexpression of CCN1 conferred resistant to carboplatin-induced apoptosis in SKOV3 cells. CONCLUSIONS: This is the first study to show abnormalities in CCN expression in ovarian carcinomas. Furthermore, our results suggest that CCN1 may play a role in ovarian carcinogenesis by stimulating survival and antiapoptotic signaling pathways.

Adult↗
PubMed1998

Diagnostic implications of albumin messenger RNA detection and cytokeratin pattern in benign hepatic lesions and biliary cystadenocarcinoma.

Cytokeratin (CK) patterns and albumin messenger RNA (mRNA) are investigated in 24 patients with benign hepatic lesions (7 patients with focal nodular hyperplasia [FNH], 10 with hepatocellular adenomas [HA], 1 with biliary hamartoma, 4 with biliary cysts, 2 with cystadenomas) and in 8 patients with cystadenocarcinoma, a rare liver malignancy. The lesions and surrounding tissue of the hepatocytic components expressed CK 8 and 18 at immunohistochemistry, whereas the biliary elements evidenced CK 8 and 18 and CK 7 and 19. The albumin mRNA, as detected by in situ hybridization (ISH), revealed different distributions in the hepatocytes of FNH and HA. In the benign biliary lesions, the normal hepatocytes surrounding the tumors expressed albumin mRNA, whereas the biliary structures did not. Interestingly, in the cystadenocarcinomas, albumin mRNA was observed not only in the hepatocytes of residual parenchyma, but also in neoplastic bile duct cells lining the carcinomatous cysts; no signal was identified in the nonneoplastic biliary elements. This indicates that cystadenocarcinomas have a mixed biological phenotype and suggests they could arise either from pluripotent cells or from neoplastic cells that reacquire epigenetic features. Our results suggest two possible diagnostic applications for albumin ISH: on routine sections, it could represent an important tool for distinguishing between cystadenoma and cystadenocarcinoma; and on fine needle biopsy specimens, it could reduce uncertainty between FNH and HA.

Adenoma↗
PubMed2002

Expression of voltage-gated K+ channels in human atrium.

Voltage-gated K(+) channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K(+) channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K(+) channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K(+) channel mRNAs were Kv4.3 (80.7 +/- 10.1 %), Kv1.5 (69.7 +/- 11.2 %) and HERG (55.9 +/- 21.5 %). Significant expression of KvLQT1 (33.5 +/- 5.5 %,) and Kv1.4 (26.7 +/- 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 degrees C. These recordings demonstrated the existence of transient (I(to1)) and sustained (I(so)) outward currents in isolated human atrial myocytes. I(to1), and not I(so), showed voltage-dependent inactivation during 100 ms pre-pulses. Both I(to1) and I(so) were inhibited by high concentrations (2 mM) of the K(+) channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 microM) inhibited I(so) selectively. I(to1) recovered from inactivation relatively rapidly (tao approximately 21 ms). These data, with published information regarding the properties of expressed K(+) channels, suggest that Kv4.3 represents the predominant K(+) channel subunit underlying I(to1) with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation.

4-Aminopyridine↗
PubMed2000

Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols.

To identify genes that are transcriptionally activated when human macrophages accumulate excess lipids, we employed the mRNA differential display technique using RNA isolated from human monocyte-macrophages incubated in the absence or presence of acetylated low density lipoprotein and sterols (cholesterol and 25-hydroxycholesterol). These studies identified a mRNA whose levels were highly induced in lipid-loaded macrophages. The mRNA encoded the human White protein, a member of the ATP-binding cassette (ABC) transporter superfamily of proteins. The mRNA levels of ABC8, the murine homolog of the human white gene, were also induced when a murine macrophage cell line, RAW264.7, was incubated with acetylated low density lipoprotein and sterols. Additional studies demonstrated that white/ABC8 mRNA levels were induced by specific oxysterols that included 25-, 20(S)-, and 22(R)-hydroxycholesterol, and by a retinoid X receptor-specific ligand. Furthermore, the oxysterol-mediated induction of ABC8 expression in mouse peritoneal macrophages was dependent on the presence of the nuclear oxysterol receptors, liver X receptors (LXRs). Macrophages derived from mice lacking both LXRalpha and LXRbeta failed to up-regulate the expression of ABC8 following incubation with 22(R)-hydroxycholesterol. Oxysterol-dependent induction of white/ABC8 mRNA was blocked by actinomycin D but not by cycloheximide treatment of cells. We conclude that the white and ABC8 genes are primary response genes that are transcriptionally activated by specific oxysterols and that this induction is mediated by the LXR subfamily of nuclear hormone receptors. These data strongly support the hypothesis that white/ABC8 has a role in cellular sterol homeostasis.

ATP-Binding Cassette Transporters↗
PubMed2003

Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation.

Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 microm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short- or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.

Animals↗
PubMed2000

Differential expression pattern of membrane-type matrix metalloproteinases in rheumatoid arthritis.

OBJECTIVE: To study the expression of messenger RNA (mRNA) for different membrane-type matrix metalloproteinases (MT-MMPs) and compare their expression pattern in rheumatoid arthritis (RA) and normal synovium. METHODS: Polymerase chain reaction (PCR) with specific primers was performed to analyze the presence of MT1-, MT2-, MT3-, and MT4-MMP in synovial tissue and synovial fibroblasts from 10 patients with RA and 4 subjects without arthritis. In addition, in situ hybridization with digoxigenin-labeled RNA probes was used to investigate the expression pattern of MT-MMPs in the synovium of these subjects. MT-MMP-expressing cells were characterized by immunohistochemical double labeling with anti-CD68 monoclonal antibodies. RESULTS: Reverse transcription-PCR revealed the expression of MT1-, MT2-, MT3-, and MT4-MMP mRNA in all tissues and cell cultures examined. However, in situ hybridization showed considerable differences in the expression pattern of the different MT-MMPs in RA synovium. MT1- and MT3-MMP mRNA were highly expressed in both the lining and the sublining layer, with more intense staining in the lining. Immunohistochemical double labeling demonstrated the presence of mRNA for MT1-MMP in fibroblasts and macrophages, as well as in osteoclast-like cells at sites of bone resorption. Expression of MT3-MMP mRNA was seen in fibroblasts and some macrophages. Expression of MT2- and MT4-MMP was characterized by staining of only a few CD68-negative fibroblasts, and no differences could be found between the lining and sublining. Normal synovial samples showed only limited staining for all MT-MMPs. CONCLUSION: Our results indicate a role for MT1-MMP not only in the matrix degradation by fibroblasts, but also in osteoclast-mediated bone resorption in RA. Given the ability of MT1-MMP to activate MMP-2 and MMP-13, the findings also point to a cooperation between fibroblasts and macrophages in degrading cartilage and bone. While MT3-MMP is also intensely expressed in RA synovium, MT2- and MT4-MMP appear not to be involved in rheumatoid joint destruction.

Arthritis, Rheumatoid↗
PubMed1995

Novel isoforms of murine intercellular adhesion molecule-1 generated by alternative RNA splicing.

Intercellular adhesion molecule-1 (ICAM-1)-deficient mice, produced by homologous recombination and previously recognized to be devoid of the common form of ICAM-1, are shown to express residual amounts of ICAM-1 Ag in thymus and lung. We demonstrate that this expression of ICAM-1 is possible because the mutated exon 5 in these animals has been skipped by alternative splicing of RNA. Three different alternative isoforms of ICAM-1 are expressed in mutant mice. Moreover, two of these isoforms are expressed in wild-type mice together with two additional alternative isoforms that cannot be produced in mutant mice. All alternatively spliced isoforms of ICAM-1 detected are missing complete extracellular Ig domains. In both mutant and wild-type mice, expression of alternatively spliced isoforms is up-regulated following stimulation of animals with LPS. Furthermore, all alternative isoforms of ICAM-1, except one, retain the ability to bind to the well-recognized ICAM-1 counter-receptor LFA-1. These findings, along with the restricted tissue distribution in mutant mice, indicate that alternative isoforms of ICAM-1 are significant physiologic adhesion structures which could play a distinct role in the functioning of the immune system of intact animals.

Alternative Splicing↗
PubMed2006

Intrinsic and selected resistance to antibiotics binding the ribosome: analyses of Brucella 23S rrn, L4, L22, EF-Tu1, EF-Tu2, efflux and phylogenetic implications.

BACKGROUND: Brucella spp. are highly similar, having identical 16S RNA. However, they have important phenotypic differences such as differential susceptibility to antibiotics binding the ribosome. Neither the differential susceptibility nor its basis has been rigorously studied. Differences found among other conserved ribosomal loci could further define the relationships among the classical Brucella spp. RESULTS: Minimum inhibitory concentration (MIC) values of Brucella reference strains and three marine isolates to antibiotics binding the ribosome ranged from 0.032 to >256 microg/ml for the macrolides erythromycin, clarithromycin, and azithromycin and 2 to >256 microg/ml for the lincosamide, clindamycin. Though sequence polymorphisms were identified among ribosome associated loci 23S rrn, rplV, tuf-1 and tuf-2 but not rplD, they did not correlate with antibiotic resistance phenotypes. When spontaneous erythromycin resistant (eryR) mutants were examined, mutation of the peptidyl transferase center (A2058G Ec) correlated with increased resistance to both erythromycin and clindamycin. Brucella efflux was examined as an alternative antibiotic resistance mechanism by use of the inhibitor L-phenylalanine-L-arginine beta-naphthylamide (PAbetaN). Erythromycin MIC values of reference and all eryR strains, except the B. suis eryR mutants, were lowered variably by PAbetaN. A phylogenetic tree based on concatenated ribosomal associated loci supported separate evolutionary paths for B. abortus, B. melitensis, and B. suis/B. canis, clustering marine Brucella and B. neotomae with B. melitensis. Though Brucella ovis was clustered with B. abortus, the bootstrap value was low. CONCLUSION: Polymorphisms among ribosomal loci from the reference Brucella do not correlate with their highly differential susceptibility to erythromycin. Efflux plays an important role in Brucella sensitivity to erythromycin. Polymorphisms identified among ribosome associated loci construct a robust phylogenetic tree supporting classical Brucella spp. designations.

Anti-Bacterial Agents↗