Trh Genes Research Articles

RXR acts as a coregulator in the regulation of genes of the hypothalamo-pituitary axis by thyroid hormone receptors.

Thyroid hormone receptors (TRs) often modulate transcriptional activity of target genes by heterodimerization with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. RXR action on negative TREs is unclear. Furthermore, the single half-site configuration of many negative TREs does not favor the binding of a classic TR-RXR heterodimer. In a comparative study using CV-1 cells (relatively RXR- and TR-deficient) and JEG-3 cells (relatively TR-deficient), we demonstrate the importance of RXR in the negative transcriptional regulation of genes of the hypothalamo-pituitary axis by tri-iodothyronine. While RXR has variable effects on ligand-independent activation produced by TRs, it was required for efficient ligand-dependent repression of the TRH gene for TRalpha1 and TRbeta1 and of the TSH genes by all TRs. Using different RXR constructs we also observed the importance of the C-terminus of RXR but not of the N-terminus nor the DNA-binding domain, in the potentiation of negative regulation. We thus suggest that, with regard to negative regulation of the TRH and TSH genes by thyroid hormones, RXR behaves more like a cofactor than a classic heterodimerization partner.

Occurrence of pathogenic vibrios in coastal areas of France.

This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.

Intracerebroventricular administration of α-melanocyte stimulating hormone increases phosphorylation of CREB in TRH- and CRH-producing neurons of the hypothalamic paraventricular nucleus

Changes in circulating leptin levels, as determined by nutritional status, are important for the central regulation of neuroendocrine axes. Among these effects, fasting reduces TRH gene expression selectively in the hypothalamic paraventricular nucleus (PVN), which can be reversed by leptin administration. Intracerebroventricular (i.c.v.) infusion of α-MSH recapitulates the effects of leptin on hypophysiotropic TRH neurons, completely restoring proTRH mRNA to levels in fed animals despite continuation of the fast, making α-MSH a candidate for mediating the central effects of leptin. As α-MSH binds to a G-protein coupled receptor that activates cAMP and α-MSH stimulates the TRH promoter through the phosphorylation of the transcription factor CREB in vitro, we determined whether i.c.v. injection of α-MSH to rats regulates phosphorylation of CREB, specifically in hypophysiotropic TRH neurons of PVN. As α-MSH also induces the activation of CRH gene expression in the PVN, we further determined whether i.c.v. injection of α-MSH regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. In vehicle-treated animals, only rare neurons contained nuclear phospho-CREB (PCREB) immunoreactivity in the parvocellular PVN. I.c.v. injection of 10 μg α-MSH dramatically increased the number of PCREB-immunolabeled cell nuclei in the PVN in fasted groups at 10 min postinjection, particularly in the medial, periventricular, anterior and ventral parvocellular subdivisions, whereas a moderate increase of PCREB immunoreactivity was observed at 30 min and PCREB immunoreactivity was lowest at 1 h postinfusion. Double immunolabeling with proTRH antiserum revealed that following i.c.v. α-MSH infusion at 10 min, the majority of TRH neurons contained PCREB in the anterior (71%), medial (83%) and periventricular (63%) parvocellular subdivisions. The percentage of double-labeled TRH neurons declined at 30 min and 1 h post α-MSH infusion. Similarly, only 16% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle treated animals, whereas 10 min following α-MSH infusion the percentage of CRH neurons colocalizing with the PCREB rose to 54%, then fell to 37 and 17% at 30 and 60 min postinfusion, respectively. These data demonstrate that i.c.v. α-MSH administration increases the phosphorylation of CREB in several subdivisions of the PVN including TRH and CRH neurons in the medial and periventricular parvocellular subdivisions, suggesting that phosphorylation of CREB may be necessary for α-MSH-induced activation of the TRH and CRH genes. The increase in PCREB in the anterior and ventral parvocellular subdivisions of the PVN, regions linked to nonhypophysiotropic functions such as autonomic regulation, would also imply a role for these neurons in anorectic and energy wasting responses of melanocortin signaling.

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS–PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40°C to 50°C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan

Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.

Abundance of Vibrio parahaemolyticus having tdh and/or trh genes in an area of Seto-Inland Sea, Japan.

Abundance of Vibrio parahaemolyticus having tdh and/or trh genes in an area of Seto-Inland Sea, Japan.

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3[ratio ]K6 and O4[ratio ]K68) in Thailand and India

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses.

Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the tdh gene but not the trh gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150-3155, 1997). Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the tdh and trh typing, and AP-PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the toxRS sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus Vibrio. The toxRS sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed. This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method. The tdh-positive and trh-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their toxRS sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of V. parahaemolyticus and reports a novel toxRS-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.

Regulation of the Mouse Preprothyrotropin-Releasing Hormone Gene by Retinoic Acid Receptor

Retinoic acid (RA) has been reported to inhibit the secretion and synthesis of the pituitary TSH in vivo and in vitro. However, little is known about the influence of RA on the expression of the prepro-TRH gene. We therefore investigated whether the promoter activity of the mouse TRH gene is directly regulated by RA using a transient transfection assay into CV-1 cells. In the absence of cotransfected RA receptor (RAR), all-trans-RA did not affect the promoter activity. In contrast, the cotransfected RARα significantly stimulated promoter activity in the absence of ligand, and all-trans-RA reversed basal promoter activation. The cotransfected thyroid hormone receptor-β (TRβ), but not 9-cis-RA receptor (RXR), had an additive effect on the RAR-dependent stimulation. TR and RAR can similarly interact with the corepressor proteins, and the cotransfected nuclear receptor corepressor (N-CoR) has been demonstrated to augment the transcriptional stimulation of the TRH gene by unliganded TR. As observed with TR, the coexpression of a N-CoR variant significantly enhanced the ligand-independent stimulation by RAR. A mutant RAR (RAR403) lacking the C-terminal activation function-2 (AF-2) activation domain that was essential for ligand-induced corepressor release constitutively stimulated the promoter activity. The constitutive stimulation by RAR403 was augmented by the cotransfected N-CoR variant. A deletion analysis of the 5′-flanking region of the TRH gene revealed that the minimal promoter region for the regulation by RAR was −83 to+ 53, with a consensus half-site motif for the thyroid hormone response element at −57. In contrast to the strong binding of TR to the thyroid hormone response element half-site in gel retardation assays, no binding of RAR homodimer, RAR/RXR heterodimer, or RAR/TR heterodimer was observed to the minimal promoter region. These results collectively suggest that RAR without heterodimerization with RXR and TR regulates transcription of the mouse TRH gene in cooperation with the corepressor, and that the DNA binding of RAR appeared to be unnecessary for regulation of the TRH gene promoter.

Regulation of the mouse preprothyrotropin-releasing hormone gene by retinoic acid receptor.

Retinoic acid (RA) has been reported to inhibit the secretion and synthesis of the pituitary TSH in vivo and in vitro. However, little is known about the influence of RA on the expression of the prepro-TRH gene. We therefore investigated whether the promoter activity of the mouse TRH gene is directly regulated by RA using a transient transfection assay into CV-1 cells. In the absence of cotransfected RA receptor (RAR), all-trans-RA did not affect the promoter activity. In contrast, the cotransfected RARalpha significantly stimulated promoter activity in the absence of ligand, and all-trans-RA reversed basal promoter activation. The cotransfected thyroid hormone receptor-beta (TRbeta), but not 9-cis-RA receptor (RXR), had an additive effect on the RAR-dependent stimulation. TR and RAR can similarly interact with the corepressor proteins, and the cotransfected nuclear receptor corepressor (N-CoR) has been demonstrated to augment the transcriptional stimulation of the TRH gene by unliganded TR. As observed with TR, the coexpression of a N-CoR variant significantly enhanced the ligand-independent stimulation by RAR. A mutant RAR (RAR403) lacking the C-terminal activation function-2 (AF-2) activation domain that was essential for ligand-induced corepressor release constitutively stimulated the promoter activity. The constitutive stimulation by RAR403 was augmented by the cotransfected N-CoR variant. A deletion analysis of the 5'-flanking region of the TRH gene revealed that the minimal promoter region for the regulation by RAR was -83 to +53, with a consensus half-site motif for the thyroid hormone response element at -57. In contrast to the strong binding of TR to the thyroid hormone response element half-site in gel retardation assays, no binding of RAR homodimer, RAR/ RXR heterodimer, or RAR/TR heterodimer was observed to the minimal promoter region. These results collectively suggest that RAR without heterodimerization with RXR and TR regulates transcription of the mouse TRH gene in cooperation with the corepressor, and that the DNA binding of RAR appeared to be unnecessary for regulation of the TRH gene promoter.

Embryonic silk gland development in Bombyx: molecular cloning and expression of the Bombyx trachealess gene.

We describe embryonic development of the Bombyx silk gland. To extend the analysis further we isolated a Bombyx counterpart gene of the Drosophila trachealess (trh) gene. Bombyx trh encodes a protein of 849 amino acids. When compared with the amino acid sequence of Drosophila trh, the identity of Bombyx bHLH, PAS-A and PAS-B domains is 100%, 97%, and 80%, respectively. Northern blot analysis revealed the presence of a single Bombyx trh transcript of 5.4 kb. We analyzed the expression pattern of the Bombyx trh transcript during embryogenesis by in situ hybridization. Bombyx trh mRNA was first detected in the tracheal primordial cells at around embryonic stage 18. Thereafter levels of Bombyx trh mRNA increased, and the high expression level was maintained until hatching. At embryonic stage 19 the transcript was also detected in the posterior basal region of the labial segment from where the silk gland invaginates. By the blastokinesis stage (around stage 23), the silk gland was lengthened, and, interestingly, the Bombyx trh transcript was restricted to the anterior silk gland. These results suggest that Bombyx trh plays a role in the formation of the trachea and the anterior silk glands.

Evidence from studies with N-ethyl-maleimide and 12- O-tetradecanoylphorbol-13-acetate that AP-1 and CREB are involved in the glucocorticoid activation of TRH gene expression in hypothalamic cultures

To determine whether c- fos/c- jun (AP-1) and CREB mediate glucocorticoid stimulated TRH gene regulation, we investigated the effect of N-ethyl-maleimide (NEM), an alkylating agent, and 12- O-tetradecanoylphorbol-13-acetate (TPA) on this process. NEM decreased while TPA increased TRH levels in rat hypothalamic culture, changes similar to their effects on CREB and Fos/Jun proteins in the AtT 20 cell line. This suggests that glucocorticoid stimulation of TRH gene expression may be regulated by the AP-1 complex and CREB pathway.

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V. parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10 1–10 2 cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10 4 cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.

Cloning of Thyrotropin-Releasing Hormone Precursor and Receptor in Rat Thymus, Adrenal Gland, and Testis

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Cloning of thyrotropin-releasing hormone precursor and receptor in rat thymus, adrenal gland, and testis.

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Feedback Regulation of Thyrotropin-Releasing Hormone Gene Expression by Thyroid Hormone in the Caudal Raphe Nuclei in Rats**This work was supported by NIH Grants DK-50255 (H.Y.), DK-30110 (Y.T.), and DK-41301, Pilot and Feasibility Award (to H.Y.).

Medullary TRH regulates autonomic activity, and altered thyroid status is associated with autonomic disorders. We investigated whether thyroid hormone exerts a negative feedback regulation on TRH gene expression in the medullary caudal raphe nuclei. Medullary pro-TRH messenger RNAs (mRNAs) were mainly located in the raphe pallidus and raphe obscurus neurons as shown by in situ hybridization and were significantly increased by 70% and 160-230% by Northern blot analyses in 24 h fasted rats at 1 and 3-5 weeks after thyroidectomy, respectively, when serum T4 levels were reduced by 75-87%. The increased pro-TRH mRNA on the 30th day after thyroidectomy was reversed to euthyroid levels by T4 replacement (2 or 4 microg/100 g x day). T4 injections (10 or 100 microg/100 g x day for 30 days) did not significantly influence medullary pro-TRH mRNA levels in sham-operated rats. Thyroidectomized rats fed normally showed a 500% increase in pro-TRH mRNA levels 30 days after the surgery, while those fasted for 24 h showed only a 180% increase. These data indicate that medullary TRH gene expression is enhanced during hypothyroidism due to the lack of negative feedback regulation by thyroid hormone, and this response is modulated by feeding state. These findings may have important relevance to understanding autonomic-related visceral alterations induced by hypothyroidism.

Close proximity of the tdh, trh and ure genes on the chromosome of Vibrio parahaemolyticus.

The distribution and location of the virulence-factor genes of Vibrio parahaemolyticus, tdh and trh, and the structural gene of urease, ureC, were examined on the genomic DNAs of 115 clinical isolates of V. parahaemolyticus. The majority of strains (81%) had two copies of tdh on the chromosome, and no copies of trh or ure. Southern hybridization with a tdh probe, after pulsed-field gel electrophoresis of Notl-digested genomic DNA of each strain revealed only single bands, suggesting that the two copies of the exist on single Notl fragments in each strain. Of the 115 strains, 7% had the tdh, trh and ure genes on chromosomal DNA. The three genes were also detected on single Notl fragments in these strains. More detailed analysis revealed that the three genes were localized within 40 kb. By long and accurate polymerase chain reactions (LA-PCR) the distance between trh and ure was shown to be less than 8.5 kb. These results reveal a close proximity of the tdh, trh and ure genes on the chromosome of pathogenic V. parahaemolyticus strains.

Intracisternal antisense oligodeoxynucleotides to the thyrotropin-releasing hormone receptor blocked vagal-dependent stimulation of gastric emptying induced by acute cold in rats.

Cold exposure increases TRH gene expression in hypothalamic and raphe nuclei and results in a vagal activation of gastric function. We investigated the role of medullary TRH receptors in cold (4-6 C, 90 min)-induced stimulation of gastric motor function in fasted conscious rats using intracisternal injections of TRH receptor (TRHr) antisense oligodeoxynucleotides (100 microg twice, -48 and -24 h). The gastric emptying of a methyl-cellulose solution was assessed by the phenol red method. TRH (0.1 microg) or the somatostatin subtype 5-preferring analog, BIM-23052 (1 microg), injected intracisternally increased basal gastric emptying by 34% and 47%, respectively. TRHr antisense, which had no effect on basal emptying, blocked TRH action but did not influence that of BIM-23052. Cold exposure increased gastric emptying by 64%, and the response was inhibited by vagotomy, atropine (0.1 mg/kg, i.p.), and TRHr antisense (intracisternally). Saline or mismatched oligodeoxynucleotides, injected intracisternally under similar conditions, did not alter the enhanced gastric emptying induced by cold or intracisternal injection of TRH or BIM-23052. These results indicate that TRH receptor activation in the brain stem mediates acute cold-induced vagal cholinergic stimulation of gastric transit, and that medullary TRH may play a role in the autonomic visceral responses to acute cold.

Arcuate Nucleus Ablation Prevents Fasting-Induced Suppression of ProTRH mRNA in the Hypothalamic Paraventricular Nucleus

Fasting results in reduced thyroid hormone levels and inappropriately low or normal thyroid-stimulating hormone (TSH), partly attributed to central hypothyroidism due to suppression of pro TRH gene expression in the hypothalamic paraventricular nucleus. Recently, we demonstrated that the systemic administration of leptin to fasting animals restores plasma thyroxine (T₄) and proTRH mRNA in the paraventricular nucleus to normal, suggesting that the fall in circulating leptin levels during fasting acts as a signal to hypophysiotropic neurons in the paraventricular nucleus to reset the set point for feedback regulation of pro TRH mRNA by thyroid hormone. To determine whether the effect of fasting on the hypothalamic-pituitary-thyroid axis is mediated through the hypothalamic arcuate nucleus where leptin receptors are highly concentrated, we studied the effect of fasting and exogenous leptin administration on plasma thyroid hormone levels and proTRH mRNA concentration in the paraventricular nucleus in adult animals with arcuate nucleus lesions induced pharmacologically by the neonatal administration of monosodium L-glutamate (MSG). In normal animals, fasting reduced plasma T₄ and TSH levels and the concentration of proTRH mRNA in the hypothalamic paraventricular nucleus. In contrast, neither fasting nor leptin administration to fasting MSG-treated animals had any significant effects on plasma thyroid hormone and TSH levels and proTRH mRNA in the paraventricular nucleus. These studies suggest that during fasting, the arcuate nucleus is essential for the normal homeostatic response of the hypothalamic-pituitary-thyroid axis and may serve as a critical locus to mediate the central actions of leptin on proTRH gene expression in the paraventricular nucleus.