Pathogenic potential of Vibrio parahaemolyticus isolated from tropical estuarine environments in Ceará, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide. This research aimed to investigate the occurrence of V. parahaemolyticus in estuarine environments and determine the virulence profile in an aquaculture environment by molecular techniques and conventional microbiological methods. Sampling was conducted in four estuaries in the State of Ceará (Pacoti, Choró, Pirangi and Jaguaribe), Brazil, between January and April 2009. The analysis included 64 samples of water (n=32) and sediment (n=32) collected from the estuaries. The samples yielded 64 isolates suspected to be V. parahaemolyticus. The isolates were submitted to biochemical identification using a dichotomous key and PCR for the detection of the species-specific tlh gene. Virulence was assessed by testing for urea hydrolysis and ß-hemolysis in erythrocytes (Kanagawa phenomenon) and simultaneous detection of the tdh and trh genes. All but one of the isolates (63/64) were confirmed to be V. parahaemolyticus by genotypic detection of tlh gene. The tdhand trh genes were detected in 57 and 19 isolates, respectively. The Kanagawa test was positive for 51 isolates. Only one isolate was positive for urease. The incidence of tdh/trh-positivity was very high in isolates recovered from the environment. The present study demonstrates the need to increase knowledge of the ecology and pathogeny of V. parahaemolyticus

Virulence gene detection and multi-locus sequence typing of Vibrio parahaemolyticus from patients in Ningbo,China / 中国人兽共患病学报

To investigated the toxin genes distribution and molecular characteristics of Vibrio parahaemolyticus from pa‐tients in Ningbo ,V .parahaemolyticus strains were collected from patients with food poisoning and diarrhea .Thermostable di‐rect hemolysin gene (tdh) and TDH‐related hemolysin gene (trh) were detected by polymerase chain reaction (PCR) .Molecu‐lar characteristics were acquired by multi‐locus sequence typing (MLST ) .Of 248 clinical strains were isolated from 2006 to 2012 .Forty‐eight strains were selected to detect virulence genes and MLST genotyping .Forty‐two isolates were detected as tdh+ and 11 isolates were detected as trh+ .There were 9 STs and one undifferentiated type in Ningbo clinical strains .Thirty‐two strains were classified into ST3 ,5 strains into ST265 and 3 strains into ST120 .ST265 was found in Ningbo strains com‐pared with strains from other regions of China .Strains with tdh+ accounted for the majority in Ningbo clinical strains .Twen‐ty‐five strains of ST3 clone were tdh+ /trh‐.There were 9 STs coexsited in Ningbo clinical strains .ST3 clone was dominant , followed by ST265 and ST120 .Strains with tdh+ /trh‐were dominant in the ST3 clone .The unique ST262 was found in Ning‐bo clinical strains .

La hormona liberadora de tirotropina (TRH) del núcleo paraventricular hipotalámico y sistema límbico como reguladora de la homeostasis energética y de la conducta alimentaria en animales con ayuno, restricción alimentaria y anorexia / Thyrotropin-releasing hormone from the hypothalamic paraventricular nucleus and limbic system regulates energy homeostasis and feeding behavior in fasted, food retricted and anorexic animals

TRH expression and release from hypothalamic paraventricular nucleus (PVN) change with environmental stimuli. Fasted and food-restricted animals present decreased TRH synthesis and release, decelerating metabolic rate and utilization of energy stores, which is an advantageous adaptation of animals with nutrient deficit. Comparing thyroid axis function between prepuberal vs. adult male fasted animals, we found a greater body weight reduction than in adults (30% vs.11%) and TRH release was not decreased; TRH degradation by pituitary PPII enzyme decreased, which maintained energy waste. TRH content of fasted-prepuberal animals changed in hippocampus and nucleus accumbens, and in amygdala of adults vs. ad libitum fed animals. PVN TRH role in food-avoiding behavior was studied by comparing its expression levels and of adolescent, adult females and male animals with anorexic conduct when drinking 2.5% of NaCl solution (AN) vs. a group forced to ingest the amount of food consumed by AN (FFR); also vs. a control group fed ad libitum (C). PVN TRH mRNA and TSH serum levels increased in AN vs. C; both decreased in FFR, supporting the putative anorexigenic role for the peptide. TRH content differentially changed in hippocampus and in frontal cortex of AN and FFR, suggesting its participation in taste perception and memory association. Orexinergic and NPYergic pathways are inactive in anorexic animals. Blocking corticotrophin-releasing hormone signal by an antagonist of CRH-R2 in the PVN reverses TRH high expression and TSH serum levels in AN.

La expresión y liberación de la TRH del núcleo paraventicular hipotalámico (NPV) cambia con estímulos ambientales; en ayuno y restricción de alimentos la liberación del péptido disminuye, reduciéndose la tasa del metabolismo y la degradación de reservas energéticas. Esto es una adaptación ventajosa para los animales con balance negativo de energía. Al comparar el contenido de TRH en la eminencia media entre animales prepúberes y adultos en ayuno de 48 horas, observamos que los jóvenes no tienen una adaptación al déficit de nutrimentos. Su peso baja más que en adultos (30% vs. 11%) y la liberación de TRH no disminuye; la degradación de TRH por PPII en la adenohipófisis (PPII) disminuye, manteniéndose el gasto energético. El contenido de TRH de animales prepúberes en ayuno cambió en el hipocampo y en el núcleo accumbens, así como en la amígdala de los adultos comparado contra los animales con alimentación ad libitum. La TRH se ha propuesto como agente anorexigénico. Evaluamos su contenido y expresión en el NPV de animales que evitan el alimento al beber una solución de NaCl (2.5%)(AN), en otros con restricción de alimento forzada (RAF) que ingieren la misma cantidad que AN y en aquéllos (C) con alimentación ad libitum. La síntesis de TRH en el NPV y el contenido sérico de TSH disminuyen en RAF pero aumentan en AN. La vía orexinérgica y la de NPY de AN están inactivas. La inyección de un antagonista a CRH revierte las alteraciones de TRH y TSH y atenúa la anorexia de AN.

Avaliação do leucograma de mulheres pós-menopausa residentes no município de Catuípe-RS, Brasil / Evalluation of leukogran of postmenopausal women living in the city of Catuípe-RS, Brazil

Os hormônios esteróides, tanto andrógenos quanto estrógenos, afetam o desenvolvimento e o comportamento sexual e uma variedade de outras funções reprodutoras. A fim de avaliar a interferência hormonal no organismo feminino, desenvolveu-se este estudo em mulheres pós-menopausa, com no mínimo um ano de amenorreia, residentes na área urbana do município de Catuípe/RS. A análise do leucograma permitiu verificar alterações hematológicas, determinando possíveis interferências no sistema imune de mulheres. Além disso fez-se referência ao uso, ou não, de Terapia de Reposição Hormonal (TRH) pelas pacientes, a fim de demonstrar a influência hormonal neste período de vida feminino. De acordo com o estudo realizado nestas mulheres pós-menopausa, níveis baixos de estrogênio mostraram atenuar a resposta imune e predispor o organismo à invasão microbiana e infecções. Assim, apesar da necessidade de pesquisas mais aprofundadas comprovando os resultados obtidos, acredita-se que a TRH melhora ou resgata a resposta imune celular afetada após a menopausa, uma vez que os esteroides sexuais têm um papel importante na regulação do sistema imune.

"Fito-hormônios": ciência e natureza no tratamento do climatério / "Phyto-hormones": Science and nature in the treatment of menopause

Um novo produto chamado "fito-hormônio" foi elaborado para substituir a terapia de reposição hormonal (TRH) nos últimos anos. Este desenvolvimento está associado a dois contextos históricos, a saber: o anúncio dos riscos associados à terapia de reposição hormonal (TRH) para mulheres no climatério no ano de 2002, e uma mudança de percepção do público consumidor e laboratórios farmacêuticos a respeito das plantas medicinais. Algumas plantas já utilizadas por comunidades tradicionais para sintomas associados ao climatério foram pesquisadas cientificamente nas últimas décadas e, como resultado, uma nova categoria de medicamento foi constituída. A partir das teorias ator-rede e de translação, de Bruno Latour, analisa-se a construção desta categoria de medicamento, e como a ideia de "natural" foi ressignificada pelos atores envolvidos na rede sócio-técnica.

A new product called "phyto-hormone" was designed to replace the hormone replacement therapy (HRT) in recent years. This development is associated with two historical contexts: the announcement of the risks associated with hormone replacement therapy (HRT) for postmenopausal women in 2002, and a change in public perception of consumers and pharmaceutical companies about medicinal plants. Some plants used by traditional communities for symptoms associated with menopause have been scientifically researched in recent decades and, as a result, a new category of medicine was established. From the actor-network theory and translation, Bruno Latour analyzes the construction of this class of drug, and how the idea of "natural" was re-signified by the actors involved in the socio-technical network.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil / Caracterização de Vibrio parahaemolyticus isolados de ostras e mexilhões em São Paulo, Brasil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100 percent of isolates, the tdh gene was identified in two (10.5 percent) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Vibrio parahaemolyticus é uma bactéria marinha, responsável por gastroenterite em humanos. A maioria dos isolados clínicos produzem hemolisina termoestável direta (TDH) e hemolisina TDH-relacionada (TRH) codificadas por genes tdh e trh, respectivamente. Neste estudo, vinte e três V. parahaemolyticus, previamente isolados de ostras e mexilhões foram analisados por PCR utilizando indicadores específicos para o gene 16S rRNA, genes de virulência (tdh, trh e tlh), resistência a diferentes classes de antibióticos, e PFGE. Dezenove isolados foram confirmados por PCR, como V. parahaemolyticus. O gene tlh estava presente em 100 por cento dos isolados, o gene tdh foi identificado em dois (10,5 por cento) dos isolados, enquanto que o gene trh não foi detectado. Cada isolado foi resistente a pelo menos um dos nove antibióticos testados. Além disso, todos os isolados apresentaram resultado positivo para o gene blaTEM-116. A presença deste gene em V. parahaemolyticus indica a possibilidade de propagação desse gene no ambiente. Cepas atípicas de V. parahaemolyticus foram também detectadas neste estudo.

Acceleration of the Increase of Endogenous Thyrotropin Hormone for Follow up Studies or Radioiodine Treatment in Patients with Differentiated Thyroid Carcinoma / Incremento rápido de tirotrofina endógena en pacientes con carcinoma diferenciado de tiroides para seguimiento o tratamiento con radioyodo

In follow up (F-U), ablation (A), or treatment (T) with radioiodine of patients with differentiated thyroid carcinoma (DTC), it is necessary to obtain elevated figures of serum TSH to assess hTg serum values or carry out 131I scanning. During the past few decades, the method employed was the withdrawal of hormonal treatment (WTH) for several weeks and its variants with the inconvenient symptoms of hypothyroidism, often restraining the use of this method. We aimed to obtain a rapid rice of serum TSH after a very short withdrawal of thyroid hormonal treatment (eight to nine days ) with the use of three or four intravenous application of TRH (200 mcg) during the first 6 days of withdrawal (TRH-St). One hundred determinations were carried out in 66 patients with DTC (ages19-80 y.o ), 20 males and 46 females. Sixty seven TRH-St were carried out for F-U, 20 for FU/T and 13 for A. In all cases the TSH values after the 3rd or 4th TRH application (samples 1 and 2) were over the value of 25 mIU/L and in the case of the second sample 99/100 determination were over the value of 30 mU/L. The values obtained were for the first sample 70.9 mIU/L ± 54.5 (range 25-310) and for the second sample 85.2 ± 61.3 (range 26-360), p<0.001. Patients considered that the symptoms and discomfort observed were mild when compared to those observed in patients submitted previously to the WTH method for 4/5 weeks. The results observed with TRH-St, allow us to consider the method as an alternative to the classic withdrawal method or the use of rhTSH with an adequate relation cost benefit.

Para efectuar ablación (A) , tratamiento con radioyodo (T) o seguimiento (S) en pacientes portadores de carcinoma diferenciado de tiroides (CDT) se hace necesario incrementar los valores de tirotrofina sérica (TSH) para elevar la sensibilidad del centellograma y la especificidad de la determinación de tiroglobulina sérica (hTg). Por años el método clásico fue la suspensión del tratamiento opoterápico (WTH) o sus variantes y ocasionalmente el uso de TSH de origen animal o , raramente, humana. Hace una década, la introducción de la TRH recombinante (rhTSH) significó evitar la desagradable sintomatología del hipotiroidismo que conllevaba el uso del método (WTH) y que en ocasiones impedía su utilización. Nuestro objetivo: el rápido ascenso de la TSH sérica después de muy breve WTH (ocho a nueve días) utilizando tres o cuatro aplicaciones intravenosas de la hormona liberadora de tirotrofina (TRH) durante los primeros seis días de WTH, método que denominamos TRH-St. Se efectuaron cien TRH-St en 66 pacientes: 20 masculinos, 46 femeninos, edades 19-80 años; 61 carcinomas papilares de diversas variantes anatomopatológicas, 4 foliculares y una variantes Hürthle. En todos los estudios después de la 3ra y cuarta aplicación de TRH (muestras 1 y 2 respectivamente) los valores de TSH fueron superiores a 25 mUI/L y con respecto a la cuarta TRH, 99/100 estudios ofrecieron valores de TSH superiores a 30 mUI/L. Los promedios obtenidos fueron: muestra 1 : 70.9 ± 54,5 mUI/L de TSH (rango 25-310); muestra 2: 85.2 ± 61.3 (rango 26-360): p < 0,001. Los pacientes consideraron que la sintomatología adversa del hipotiroidismo y el "disconformismo" fueron leves y sin comparación con los observados por aquellos pacientes sometidos anteriormente al método de supresión hormonal por 4/5 semanas.. Estas observaciones nos llevan a considerar que el método TRH-St , es una alternativa válida del método clásico de suspensión hormonal o del uso de rhTSH con una relación adecuada costo / beneficio.

Estimulación iterativa (EI) de TSH endógena (TSH-En) mediante el uso de la hormona liberadora de tirotrofina (TRH) en pacientes portadores de carcinoma diferenciado de tiroides / Rapid iterative stimulation (IS) of endogenous tsh (En-TSH) utilizing thyrotropin releasing hormone (TRH) in patients with differentiated thyroid carcinoma (DTC)

En el CDT es indispensable elevar los valores de TSH para efectuar Tg y barrido (RCT) con 131I, debiéndose suspender la opoterapia (HT) durante 4/5 sem. con el consecuente hipotiroidismo (H) y los trastornos que conlleva. Nuestro objetivo fue incrementar en forma rápida TSH-E acortando el tiempo de abstinencia. Se efectuaron 43 estudios en 37 pacientes con CDT (G-1); de entre 19 y 78 años, 34 con forma papilar y 3 folicular de CDT, 12 de sexo masculino y 25 femenino Se consideraron 2 subgrupos, G-1A, 7 p. para ablación (A); G-1B, 36 p. para seguimiento (S) y/o tratamiento (T) entre 6 meses y 5 años poscirugía; 6 p. efectuaron dos estudios, 4 para A y S y 2 para 2 veces S. Como comparación se revisaron 41 estudios en 35 p (G-2) que efectuaron suspensión de opoterapia por 4/5 semanas, entre 18 y 81 años; 28 de sexo femenino y 7 masculino; 32 papilares y 3 foliculares; 18 para A (G-2 A) y 21 para S, primer control (G-2B); 4 p. efectuaron 2 estudios, A y S. G-1A: entre 8/10 días poscirugía se les administra TRH 200 mcg i.v los días 1, 3, 5 y 6. A los 30 min de la 3ra aplicación, determinación de TSH y RCT con 370 MBq de 99mT; a igual lapso en la 4ta aplicación determinación de TSH, Tg y antiTg y 5,55 o 7,4 GBq de 131I, para A; a los 8 días RCT con 131I. G-1B: se suspende T4 y reemplaza por T3 por 3 semanas. Se suspende T3; a las 24 horas se inicia el esquema indicado para G-1A . A la 4ta aplicación de TRH, se administra el 131I, 14,8 MBq y RCT a las 48 horas en S o la actividad terapéutica indicada para T. En ambos grupos se indicó dieta hipoyódica. Resultados: En G-1, los valores de TSH ascendieron a 26-360 UI/L; promedio 83 UI/L ± 54; G-1A : 137 ±109; G-1B 7, 62 ± 52 . Los RCT no mostraron diferencias con ambos trazadores. En G-1A todos los p presentaron remanentes tiroideos y Tg positivas. En G-1B, 21 p. mostraron RCT y Tg negativas; 7 áreas activas y Tg positivas y 8 p RCT negativos con valores elevados de Tg . En G-2, TSH, 23-170 UI/L ( 63 ± 3 UI/L) ; G-2 A: 71 ± 41 ; G-2B, 63 ± 42. Conclusiones: Estos hallazgos indican que a) la metodología propuesta es adecuada para acortar sensible-mente el tiempo de abstinencia de opoterapia y reducir la sintomatología del H que pasa desapercibida en la mayoría de los casos; b) los valores de TSH-En obtenidos son similares y aun superiores a los alcanzados por suspensión de opoterapia por lapsos prolongados; c) el empleo del RCT con 99mTc como indicador de tejido captante disminuye el uso terapéutico a ciegas de 131I al señalar casos de ausencia de concentración y permite, cuando sea necesario, obtener anticipadamente 131I para su empleo terapéutico.

In the follow up (F) of p with DTC it is necessary to obtain high figures of serum TSH for determination of serum Tg and 131I scan (WBS). For this object, he method, for a long time, was to withdrawal thyroid hormone therapy (generally l-T4) that produce hypothyroidism with the inconvenient for the p, dramatics in certain cases. Our objective was to increase TSH by IS to shortening time of L-T4 withdrawal for F, ablation (A) or treatment (T) with 131I. In 37 p. with DTC (G-1), aged 19-78 y., 34 with pap. DTC and 3 with foll. form, 25 females, 12 males, 43 studies were carried out; 6 p carried 2 studies. The group was divided in 2 sub-groups: G-1A,7 p derived for A; G-1 B 36 p. for F or T with 131I. Six p carried out 2 studies; 4 of them for A and for F and 2 realizes 2 times F. All p treated with l-T4 replaced this hormone for T3 during 3 weeks ,that was withdrawal the day before IS. In G-1A, between 8/10 days after surgery they begin IS. IS: At days 1, 3, 5 and 6, the p were injected i.v. with 200 mcg of TRH; at 30 minutes of the 3rd injec. blood TSH determination ; immediately 370 MBq of 99mT was administered and at 30 minutes a WBS was carried out. At 30 minutes of the 4th injec. blood figures of TSH, Tg and Tg-ab were determined; immediately the activity of 131I indicated for each group was given to the p; in G-1A, at 8 days and in G1-B, at 48 hours WBS were carried out. As a control group (G-2) 41 studies in 35 DTC p. that withdrawal l-T4 for 4/5 weeks, were studied, aged 18-81 years, 31 females and 4 males; 32 with pap. and 3 folli.c form; 18 for A (G-2A) and 23 for F (G-2B); 6 p carried out 2 studies. One for A and the second as the first control. In G-1, TSH values obtained were 26-360 UI/L ( 83 ± 54. In G-1A : 137 ± 109 and in G-1B 62 ± 52). The 2 tracers 131I and 99mTc-Tc, produce show similar figures. In G-1A all p present thyroid remnants and elevated Tg. In G-1B, 7 p showed positive WBS and Tg; 8 p present Tg positive and WBS negative and 21 WBS and Tg negative. In G-2, the TSH values obtained were 23-179 UI/L (63 ± 39 ); G-2A 71 ± 41 and G-2B 63 ± 42. These findings indicate that the methods is adequate to shortened the time of withdrawal of l-T4 and reduce the signs/symptoms of hypothyroidism to an acceptable status. Also allow us to considered the use of 99mTc as an indicator of existence of remnants, relapses or metastases and avoid blind use of therapeutic activities of 131I.

Menopausia: tiempo para pensarla / Menopause: time to rethink it

Se hace un recuento histórico de cómo la cultura médica occidental ha percibido la menopausia, su transformación de condición fisiológica en una enfermedad y cómo a consecuencia de nuevas publicaciones relacionadas con las terapias de reemplazo hormonal se hace necesario reconsiderar muchas actitudes y prácticas médicas que se desarrollaron en los últimos años.

This article presents a historic review about the recent ways in which medicine conceives menopause, the implications of the Women's Health Initiative Studies and the need to develop a critic attitude before adopting foreign doctrines, technologies and drug therapies

Prevalence and Molecular Genetic Characteristics of Vibrio parahaemolyticus New 03:K6 and 04:K68 / 병원감염관리

BACKGROUND: Vibrio parahaemolyticus belonged to the new 03:K6 serotype was demonstrated an unusual potential to spread and an enhanced propensity to cause infection in the worldwide. Recently, increased numbers of V. parahaemolyticus 03:K6 had been isolated in Korea, and we analyzed the prevalence and molecular genetic characteristics of V. parahaemolyticus 03:K6 isolates. METHODS: V. parahaemolyticus were isolated from clinical specimens of patients with diarrhea in Hanyang University Hospital, Seoul, Korea, from 1998 to 2005. The serovars of isolates were determined by slide agglutination test with specific antisera. All isolates were examined for the presence of tdh/trh genes. AGS-PCR method detecting the new 03:K6 clone was used in this study. We analyzed clonality of these isolates by infrequent restriction site-polymerase chain reaction (IRS-PCR). RESULTS: Thirty-six strains were isolated from 1998 to 2005. The main serotype of isolates were 03:K6 (24/36: 67%), All of them have tdh gene but not trh gene and an unique toxRS gene of the new 03:K6. The morphotypes of 03:K6 isolates show a same pattern of IRS-PCR, but can easily be differentiated from non-03:K6 and 04:K68 isolates. CONCLUSION: The 03:K6 was a main serotype of V. parahaemolyticus isolated from 1998 to 2005 and they show same molecular characteristics.

Effect of thyrotrophin-releasing hormone analogue, YM14673,on the blood-brain barrier and brain edema after head trauma in rats / 中国康复理论与实践

@#Objective To study the effect of thyrotrophin releasing hormone (TRH) analogue, YM14673, on the brain edema and blood brain barrier after brain injury in rat.Methods The model of brain injury of rats was built by Feeney's methods. The Evans blue solution had been injected i.v. into the rats before the models were made. The rats were divided into four groups: normal, treated with saline, treated with YM14673(Ⅰ:0.1mg/kg and Ⅱ:1mg/kg). The water content in brain was measured 24 h after brain injured. The concentration of Evans blue in brain tissue and blood was measured with fluorometry.Results The rats treated with saline after traumatic injury showed significantly high water content compared with normal group(P<0.01)and the water content of the left hemisphere, which was hit straightly, was higher significantly than that of the right global(P<0.01). The brain water content decreased in the rats treated with YM14673 in all global(P<0.05).There was no significant different between the treated group Ⅰ and groupⅡ.After brain injury, the concentration of Evans blue in brain tissue showed a higher level contrasted with normal group. YM14673 did not influence the concentration of Evans blue in brain tissue. Conclusion YM14673 can decrease the brain edema after brain injury but it cannot decrease the permeability of blood brain barrier.

Effect of TRH on Phospholipase D Activity in GH3 Cell / 대한내분비학회지

BACKGROUND: GH3 cells are a well characterized and widely used model used for the in vitro study of growth hormone (GH) secretion. Thyrotropin releasing hormone (TRH) binds to receptors belonging to the family of G protein-coupled receptors, and secrets both GH & prolactin. Phospholipase D (PLD) is an enzyme that hydrolyses phosphatidylcholine to yield phosphatidic acid and choline, and plays important roles in cellular proliferation and hormonal secretion. To elucidate the pathway of the action of TRH in GH3 cells, we investigated the activities of PLC and PLD in GH3 cells treated with TRH or phorbor 12-myristate 13-acetate (PMA). METHODS: GH3 cells were labeled with [3H] myristate, followed by incubation of with 0.3% ethanol, prior to before the addition of the agonists. The total lipids were extracted from the harvested cells following treatment with the agonists. The PLD activity was assessed by measuring [3H] phosphatidylethanol from the [3H] phospholipid using thin layer chromatography. RESULTS: TRH (1 muM) stimulated the PLC activity by 44-fold over that of the control values. TRH (1 microM), mastoparan (5 muM), and PMA (500 muM) for 30 minutes increased PLD activity by 1.9, 1.5 and 2.2 fold, respectively, in comparison to the controls. The PLD activities after 15, 30, 60, 120 and 240 min treatments of TRH (1 microM) were 142%, 170%, 172%, 160% and 115%, respectively. CONCLUSION: These results suggest that TRH stimulates not only the PLC activity, but also the PLD activity in GH3 cells.

Virulence Factors and Genotyping of Vibrio parahaemolyticus in Sea Water and Clinical Isolates

Six strains of Vibrio parahemolyticus isolated from diarrheal patients and the 12 strains from sea water were serotyped and analyzed for biochemical characteristics, antibiotics sensitivity and detection of toxR, gyrB, tdh, and trh genes. Arbitrarily-primed polymerase chain reaction method were performed on the 6 strains from patients and the following results were obtained. 1. The Vibrio parahemolyticus isolated from patients were belonged to 5 different serotypes: 04:K8, 04:KUT, 06: K18, 010:K71 and 03:K6, but those isolated from sea water were belonged to 5 different serotypes: O1:KUT, 02:KUT, 03:K45, 04:K37 and OUT:KUT. All strains explained have different serotypes depending on the different source, 2. Three serotype (04:K8, 04:KUT, 06:K18) isolated from patients were positive for the urease hydrolysis, whereas only one strain of serotype O1:KUT isolated from sea water was positive to the same. Furthermore, the serotype 06:K18 (1 strain) was positive for the fermentation of dulcito1. Both toxR and gyrB genes were detected from all strains isolated. 3. As for control the 2 strains of serotype 03:K6 and 6 strains isolated from patients, serotype 03:K6 were resistant to oxacillin, penicillin, vancomycin. All strains were sensitive to chloramphenicol and tetracycline yet the antibiogram type showed 6 groups from I to VI. 4. DNA probe hybridization method was used to detect genes. The trh1 was detected both from serotype 04:KUT and 06:K18 isolated from patients and the trh2 was also detected from one strain from each 010:K71 and O1:KUT isolated from patients and sea-water respectively, The tdh gene only was detected from two strains of 03:K6 isolated from patients of 1998. The tdh, trh 1 and trh2 were not detected from 7 strains out of 12 strains isolated from sea water whereas the production titer of TDH isolated from patients showed from 2048 times to 4096 times. 5. Four strains of the serotype 03:K6 isolated from Korea, India and Japan as well as 3 strains from Korean patients were tested by AP-PCR to classify serotypes. As for its result the amplicon showed the same in the 4 strains of the serotype 03:K6 whereas the four strains of different serotype from patients are so difference as to explain no inter- relations at all. The result explains that the serotype 03:K6 is the same genes regardless from where it is isolated.

Detection of Thermostable Direct Hemolysin Gene and Thermostable Direct Hemolysin-Related Hemolysin Gene of Vibrio parahaemolyticus by Polymerase Chain Reaction / 대한임상병리학회지

BACKGROUND: Vibrio parahaemolyticus is one of the most important food-borne pathogens in Korea. Although the mechanism of its pathologic effect is still not clearly understood, epidemiological studies have suggested a very strong association of thermostable direct hemolysin (TDH) and thermostable direct hemolysin-related hemolysin (TRH) with the disease. We detected the tdh gene and the trh gene of V. parahaemolyticus isolates by polymerase chain reaction (PCR). METHODS: V. parahaemolyticus strains were isolated from clinical specimens of patients with diarrhea in different geographic areas of Seoul (16 cases), Inchon (27 cases) and Kwang Joo (2 cases) in Korea between 1998 and 2000. The colonies selected were identified by using the API 20E identification strip (API system, Analytab Products, Montalieu-Vercieu, France). PCR protocols were established for specific detection of the tdh and trh genes. Oligonucleotide primers were designed based on the reported nucleotide sequence of the tdh2 gene and the trh1 gene, respectively. RESULTS: The protocols established for the tdh and trh genes could detect 400 fg (100 colony-forming units) of cellular DNA carrying the respective gene. All strains of V. parahaemolyticus isolates in this study contained the tdh gene but not the trh gene. CONCLUSIONS: The detection of the tdh gene and trh gene of V. parahaemolyticus using the PCR is an easy and rapid method.

Thyrotropin-releasing Hormone(TRH) Receptor Gene Expression in GH3 Cells Permanently Transfected with a Mutant Gs alpha Gene / 대한내분비학회지

BACKGROUND: Gs alpha gene mutation, that constitutively increases intracellular cAMP, is found in some acromegalic patients. It was demonstrated that increased intracellular cAMP levels suppress the expression of rat TRH receptor (TRH-R) mRNA. We previously demonstrated that transient expression of a mutant Gs alpha gene suppress the rat TRH-R gene expression in the cultured rat growth hormone-secreting tumor cell line (GH3), whereas TRH-R gene expression in adenomas with Gs alpha gene mutation (gsp oncogene) did not differ from that in tumors without the mutation. The discrepancy suggests the possibilities that the effect of permanent expression of mutant Gs alpha gene on TRH-R gene expression is different from that of transient expression of the mutant gene and hypothalamic hormones including TRH regulate the gene expression. METHODS: We investigated whether permanent expression of the mutant-type Gs alpha does not suppress the TRH receptor gene expression in GH3 cells, and whether TRH suppresses the gene expression by using reverse transcription-polymerase chain reaction (RT-PCR) and in vitro transcription. RESULTS: Permanent expression of a mutant-type Gs alpha increased basal cAMP levels up to 1.7-fold relative to the controls, whereas the wild-type cell line did not show increased cAMP levels. Permanent expression of a mutant-type Gs alpha increased TRH receptor mRNA level up to 2.8 fold compared with the controls. Treatment of the permanently transfected GH3 cells with TRH suppressed TRH-R gene expression more prominently compared to the wild type GH3 cells. CONCLUSION: These results suggest that permanent expression of mutant Gs alpha enhances the expression of TRH-R in GH-secreting pituitary tumors with gsp oncogene, but the gene expression may also be regulated by other factors including TRH.

The Characterization of Glucocoritcoid Response Element(GRE) on the Promoter of Thyrotropin-Releasing Hormone(TRH) Gene / 대한내분비학회지

BACKGROUND: We previously demonstrated that the promoter of rat TRH gene has GRE half site (TGTTCT) between -210 bp and -205 bp flanking with similar sequences of TPA response element (TRE), TAGTCA, at a distance of several base pairs from the GRE half site. It promps us to hypothesize that this composite GRE/TRE sequence can provide a site for interaction between glucocorticoid receptor (GR) and c-Jun. Thus, we investigated whether the composite sequence mediates transcriptional regulation induced by dexamethasone (DEX) and 12-O-tetradecanoyl phobol-13-acetate (TPA), and whether it binds GR and c-Jun. METHODS: A luciferase expressing plasmids that contain a part of rat TRH promoter including the composite sequence or their mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX increased the transcriptional activity of the plasmid containing the wild type GRE by 2.5 folds, and TPA increased the transcriptional activity by 4 folds. The simultaneous stimulation with DEX and TPA synergistically increased the transcriptional activity by 10 folds. Two mutants whose GRE half sits were altered showed no responses to DEX, and suppressed the TPA-induced or both agents-induced transcriptional activity by 50%. Two mutants whose TRE-like sites were altered suppressed the DEX-induced transcriptional activity by 20%, TPA-induced trarptional activity by 25%, and both agents-induced transcriptional activity by 50%. Gel retardation assay showed that the composite sequence fonned a complex with GR and its mutants bound to GR with remarkably less affinity. c-Jun also bound to the composite sequence to form two cornplexes with less affinity compared to the AP-1 consensus sequence. The mutants of the TRE-like sequence bound to c-Jun with a significantly lower affinity compared to that of the wild type. Simulateous binding of the composite sequence with GR and c-Jun did not form any larger complex. The complex of GR and the composite sequence was much smaller than that formed by c-Jun, suggesting that GR binds to the composite sequence as a monomer. CONCLUSION: These results suggest that the composite sequence of GRE half site and TRE-like site on the promoter of rat TRH gene provides binding sites for GR and c-Jun, which mediate the interaction between two signal transduction pathways. (J Kor Soc Endocrinol 14:265-277, 1999)

Analysis of Glucocorticoid Response Element and TPA Response Element of Rat Thyrotropin-Releasing Hormine Gene by Site-Directed Mutagenesis / 대한내분비학회지

BACKGROUND: We previously demonstrated that a GRE/TRE composite sequence, which is located between 200 bp and 220 bp relative to the transcriptional start site of rat TRH gene, is responsible for the dexamethasone (DEX)- and TPA-induced transcriptional activation, and the transcriptional activation by DEX is mediated by interaction between glucocorticoid receptor (GR) and a TRE-binding transcriptional factor such as c-Jun. However, a non-specific binding with the transciption factors can not be excluded as the mutants used in the previous report could not inhibit the binding of GR and c-Jun completely, and it remains unclear which one of the two TRE-like sequences is critical for the interaction of the two transcription factors. METHODS: Luciferase expressing plasmids that contain a part of rat TRH promoter including the composite GRE sequence or its mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX or/and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX and TPA increased the transcriptional activity of the wild type composite sequence by 3 folds and 4 folds, respectively, and the combined stimulation increased the activity by 10 folds. The mutants of which all 6 nucleotides of the GRE half site were replaced and removed almost did not bind to GR and eould not enhance the transcriptional activity at all in response to DEX. The GRE-deleted mutant bound to c-Jun with a remarkably lower affinity and showed a lower response to TPA, whereas the GRE-replaced mutant bound to c-Jun with a similar affinity and showed a similar response to TPA compared to those of the wild type. In response to the combined simulation with DEX and TPA, the mutants showed 30-40% of the trancriptional activity of the wild type. Basal transcriptional activity of all the TRE mutants was significantly lower than that of the wild type. While they almost could not bind to c-Jun, their binding affinity to GR was comparable to that of the wild type. Whereas the DEX- and TPA-induced transcriptional activity of 5 TRE mutant was 10% and 15% of that of the wild type, it responded to those agents in a similar pattern as the wild type. The 3 TRE mutant and the mutant of both TRE sites did not respond to DEX and TPA. The GRE-deleted mutant hardly formed the DNA-protein complex as did the wild type, while the GRE -replaced mutant could form the complex in a less amount with nuclear extract of HeLa celL CONCLUSION: These results suggest that GRE/TRE composite sequence of rat TRH gene specifically binds to GR and c-Jun, providing a site for interaction between the two transcription factors, and that both TRE sites play an important role in basal transcription, and that the 3 TRE site is more critical in the interaction between GRE and TRE for DEX-induced transcriptional activation. (J Kor Endocrinol 14:278-292, 1999)

Antenatal Thyrotropin-Releasing Hormone to Prevent Respiratory Distress Syndrome in Preterm

PURPOSE: The purpose of this study was to assess the efficacy of antenatal thyrotropin-releasing hormone(TRH) given to mothers at risk for preterm delivery and assess various neonatal outcomes in infants born to these mothers. METHODS: Sixty-one mothers(TRH+Dexamethasone(D):30, D:31) with preterm labor at 26-34 weeks of gestational age were randomized into a study group which received 400microgram of TRH at 8 hour interval intravenously(maximum doses of 6) along with 6 mg of D at 12 hour interval intravenously(maximum doses of 4) and into a control group that received the same regimen of D only. Among 61 mothers, 17 mothers(TRH+D:8, D:9) underwent amniocentesis to document changes in L/S ratio and lamellar body count before and after TRH and or D therapy. Thyroid function tests(TFT's) were obtained from infants born at/shortly after birth, near first week and second week of life. RESULTS: Incidences of respiratory distress syndrome(42% vs 30%) and chronic lung disease(56% vs 44%) were not statistically different between the two groups. In fact, greater risk for ventilation at 28 days was observed in infants born within 24 hours of TRH treatment. All other neonatal outcomes except pulmonary hemorrhage(0% vs. 16%) were similar between TRH+D and D groups. TFT of infants born to mothers who received the last mean duration of TRH at 178 hours were similar between the two groups. CONCLUSION: Antenatal administration of TRH in addition to D did not have any additional beneficial effects compared to giving D alone.

Discrepancy between in vitro and in vivo effect of Galphas gene mutation on the mRNA expression of TRH receptor

We investigated the effect of alpha-subunit of the stimulatory GTP-binding protein (Galphas) gene mutation on the expression of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) gene in GH3 cells and in growth hormone (GH)-secreting adenomas of acromegalic patients. In the presence of cycloheximide, forskolin and isobutylmethylxanthine, cholera toxin, and GH-releasing hormone (GBRH) decreased rat TRH-R (rTRH-R) gene expression by about 39%, 43.7%, and 46.7%, respectively. Transient expression of a vector expressing mutant-type Galphas decreased the rTRH-R gene expression by about 50% at 24 h of transfection, whereas a wild-type Galphas expression vector did not. The transcript of human TRH-R (hTRH-R) gene was detected in 6 of 8 (75%) tumors. Three of them (50%) showed the paradoxical GH response to TRH and the other three patients did not show the response. The relative expression of hTRH-R mRNA in the tumors from patients with the paradoxical response of GH to TRH did not differ from that in the tumors from patients without the paradoxical response. Direct PCR sequencing of GALPHAs gene disclosed a mutant allele and a normal allele only at codon 201 in 4 of 8 tumors. The paradoxical response to TRH was observed in 2 of 4 patients without the mutation, and 2 of 4 patients with the mutation. The hTRH-R gene expression of pituitary adenomas did not differ between the tumors without the mutation and those with mutation. The present study suggests that the expression of TRH-R gene is not likely to be a main determinant for the paradoxical response of GH to TRH, and that Galphas mutation may suppress the gene expression of TRH-R in GH-secreting adenoma. However, a certain predisposing factor(s) may play an important role in determining the expression of TRH-R.

Expression of TRH Receptor Gene in GH-Secreting Piruitary Adenomas / 대한내분비학회지

Background: To test the hypothesis that Galphas gene mutation may suppress the expression of TRH-R gene, we investigated whether hTRH-R gene expression is lower in human GH-secreting pituitary adenomas with Galphas mutation than in tumors without the mutation. Method: TRH-induced paradoxical response of GH was observed in 8 acromegalic patients. The mutation of gene was identified by direct sequencing of the genomic DNA prepared from GH-secreting pituitary adenomas. The expression of hTRHT mRNA was quantitated by RT-PCR. Results: The transcript of hTRH-R gene was detected in 6 of 8(75%) tumors. Three of these(50%) showed the paradoxical GH response to TRH and the other three patients did not show the response. The relative expression of hTRH-R mRNA in the tumors from patients with the paradoxical response of GH to TRH did not differ from that in the tumors from patients without the paradoxical response. Direct PCR sequencing of Galphas disclosed a mutant allele and a normal allele only at codon 201 in 4 of 8 tumors. The paradoxical response to TRH was observed in 2 of 4 patients without the mutation, and 2 of 4 patients with the mutation. The hTRH-R gene expression of pituitary adenomas did not differ between the tumors without the mutation and those with mutation. Conclusion: This study suggests that the expression of TRH-R gene is not likely to be a main determinant for the paradoxical response of GH to TRH, and that Galphas mutation does not seem to suppress the gene expression of TRH-R in GH secreting adenoma.