Transforming growth factor-beta1 inhibits rat prolactin promoter activity in GH4 neuroendocrine cells.

The prototypic member of the transforming growth factor beta family is TGFbeta1, which is known to be important in extracellular matrix production, cell proliferation, and cell differentiation. Specifically in the pituitary lactotroph, TGFbeta1 inhibits prolactin (PRL) peptide secretion, PRL mRNA levels, and PRL gene transcription. To further elucidate the molecular details by which TGFbeta1 modulates PRL gene transcription, we used a transient transfection approach to characterize and to map the TGFbeta1 inhibitory response element of the rat (r) PRL promoter. Here, we show that TGFbeta1 selectively inhibits basal rPRL promoter activity in GH4 cells in a dose-responsive fashion, with an IC50 of 6 pM, and that this inhibition occurs within 6 h after TGFbeta1 addition. Using a series of 5' deletion promoter mutants, the TGFbeta1 inhibitory response was found to be unaffected by deletion to position -116 and was abrogated by further deletion to -54 in the rPRL promoter. However, on the basis of data from site-specific and linker-scanning mutants of the rPRL promoter, it appears that no single element is sufficient to mediate the TGFbeta1 inhibitory effect. Sequence analysis of the -116/-54 region failed to reveal any sequence homology to previously characterized TGFbeta response elements. Finally, TGFbeta1 failed to alter significantly the endogenous levels of the cell-specific activator protein GHF-1/Pit-1, indicating that the TGFbeta1 inhibitory effect is not attributable to diminished levels of GHF-1/Pit-1. Taken together, these data indicate that the TGFbeta1 inhibitory response is more complex than previously appreciated, requiring more than one cis-acting element and not always acting via TTGG or GTCTAGAC sites.

Scottish cocaine users: wealthy snorters or delinquent smokers?

Ninety-two cocaine users were interviewed in Scotland. Most were middle-class nasal users, also used other drugs and generally gave cocaine a positive rating. One half of them had at some time used cocaine more than once a week. For some, this period lasted some months, when as much as 30 'lines' of cocaine were used per day of cocaine use. More of these heavy users reported adverse effects of cocaine than was the case for light users. Nonetheless, most heavy users had reduced their use by themselves to the point that their current cocaine use was no different from that of light users. Possible explanations for this apparently spontaneous reduction are discussed.

Placental pathologic changes of maternal vascular underperfusion in bronchopulmonary dysplasia and pulmonary hypertension.

INTRODUCTION: Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of infancy, and BPD-associated pulmonary hypertension (PH) is a serious complication that can negatively impact later childhood health. There is growing evidence that lung injury leading to BPD and PH is due to chronic fetal hypoxia-ischemia. The purpose of this study was to investigate whether placental pathologic changes of maternal vascular underperfusion (MVU) are associated with BPD, and further increased with PH. METHODS: We conducted a 5-year retrospective cohort study of premature infants born ≤28 weeks. BPD was defined as persistent oxygen requirement at 36 weeks corrected gestational age. PH was identified using a standardized algorithm of echocardiogram review. Archived placental slides underwent standardized masked histopathologic review. Logistic regression modeling was performed, taking into account important maternal and infant covariates. RESULTS: Among 283 births, 121 had MVU, of which 67 (55%) developed BPD, and 24 (20%) had PH. Among the common neonatal complications of extreme prematurity, BPD was the only outcome that was increased with MVU (P < 0.001). After adjustment for birth weight, fetal growth restriction, preeclampsia and other factors, infants with MVU were more likely to develop BPD (adjusted odds ratio = 2.6; 95% confidence interval = 1.4, 4.8). Certain MVU sublesions (fibrinoid necrosis/acute atherosis and distal villous hypoplasia/small terminal villi) were increased with PH (P < 0.001). DISCUSSION: Placental MVU may identify BPD infants who were exposed to intrauterine hypoxia-ischemia, which increases their risk for development of PH disease. CONCLUSIONS: Our findings have important implications for providing earlier and more effective therapies for BPD.

The macrolide-lincosamide-streptogramin B resistance determinant from Clostridium difficile 630 contains two erm(B) genes.

The ErmB macrolide-lincosamide-streptogramin B (MLS) resistance determinant from Clostridium difficile 630 contains two copies of an erm(B) gene, separated by a 1.34-kb direct repeat also found in an Erm(B) determinant from Clostridium perfringens. In addition, both erm(B) genes are flanked by variants of the direct repeat sequence. This genetic arrangement is novel for an ErmB MLS resistance determinant.

The clostridial mobilisable transposons.

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS(A) or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties.

The c-Jun delta-domain inhibits neuroendocrine promoter activity in a DNA sequence- and pituitary-specific manner.

The transcription and transformation activity of c-Jun is governed by a 27-amino acid regulatory motif, labeled the delta-domain, which is deleted in v-Jun. We have previously shown that c-Jun is a potent inhibitor of the rat prolactin (rPRL) promoter activity induced by either oncogenic Ras or phorbol esters. Here, we have characterized the structural and cell-specific requirements for this c-Jun inhibitory response, and we show that this c-Jun inhibitory response mapped to the rPRL footprint II repressor site, was pituitary-specific and required the c-Jun delta-domain. Moreover, alteration of any one of these features (e.g., cis-element, trans-factor, or cell-specific background) switched c-Jun to a transcriptional activator of the rPRL promoter. In HeLa nonpituitary cells, c-Jun alone activated the rPRL promoter via the most proximal GHF-1/Pit-1 binding site, footprint I, and synergized with GHF-1. Finally, recombinant GHF-1 interacted directly with c-Jun but not c-Fos proteins. These data provide important fundamental insights into the molecular mechanisms by which the c-Jun delta-domain functions as a modulatory switch and further imply that the functional role of c-Jun is dictated by cell-specific influences and the delta-domain motif.

Are we ready to predict speech development from babble in cleft lip and palate children?

The speech development of nine children with cleft lip/palate was followed longitudinally from nine months to three years of age. The results indicate speech sound development closer to the non-cleft population than previous studies. Nasal fricatives previously not extensively described in the literature may be an experimental stage of developmental babble, which spontaneously reduce. The study has added to the evidence-base for practice in one cleft unit. It may be useful to channel resources at our centre to children who at nine months may be more at risk, i.e. children with bilateral clefts and known developmental delay.

Theatre in AIDS education--a controlled study.

Theatre is now widely used in HIV and AIDS education for young people. However, research concerning its effectiveness has largely been conducted without using comparison or control groups and there is little evidence that other types of intervention are more or less effective. The present study was designed to measure the effects of a theatre production on HIV knowledge, attitudes and risk behaviour of young people compared with that of a standard health education seminar which used group work techniques. Both interventions were designed for young people between the ages of 12 and 18 and held in 10 youth projects in socially deprived areas of Glasgow. Five youth projects were randomly assigned to each intervention. All participants were asked to complete a self-administered HIV knowledge, attitude and behavioural questionnaire immediately before and after the event and 2 months later. Research focus groups were also used. A total of 35 young people attending the standard seminars completed three self-administered questionnaires, and 18 participated in all three focus groups. Forty-two young people attending the theatre performances completed three self-administered questionnaires and 16 participated in all three focus groups. There was little impact on the knowledge and attitudes of either audience, although there was some evidence of attitudinal change among those exposed to the health education discussion session. More of the theatre group reported a behavioural change 2 months after attending the play but only in relation to buying and carrying condoms. It is uncertain whether this led to safer sex practices. This study does not support the view that theatre in AIDS education has a significant impact on HIV knowledge, attitudes and risk behaviour.

Metabolism of N-methylformamide in mice: primary kinetic deuterium isotope effect and identification of S-(N-methylcarbamoyl)glutathione as a metabolite.

S-(N-Methylcarbamoyl)glutathione has been identified by cesium ion liquid secondary ion mass spectrometry as a biliary metabolite in mice of the experimental antitumor agent and hepatotoxin N-methylformamide. Metabolism of N-methylformamide to urinary methylamine, urinary N-acetyl-S-(N-methylcarbamoyl)-cysteine and biliary S-(N-methylcarbamoyl)glutathione was found to be subject to large intermolecular primary kinetic isotope effects when hydrogen was replaced by deuterium in the formyl group (kH/kD = 5.5 +/- 0.2, 4.5 +/- 1.0 and 7 +/- 2, respectively), as shown by mass spectrometry of derivatives of these metabolites. These values indicate the existence of a common metabolic precursor for each of these metabolites. In particular, methylamine is shown not to arise from simple enzymatic hydrolysis of N-methylformamide but is associated with an oxidative process. Therefore, it is highly likely that N-methylformamide is oxidized and conjugated to form S-(N-methylcarbamoyl)glutathione which is metabolized further to N-acetyl-S-(N-methylcarbamoyl) cysteine. Either of these thiocarbamates could be hydrolyzed to give the parent thiol and the observed metabolic end products, methylamine and carbon dioxide. The presence of deuterium in the formyl moiety of N-methylformamide reduced markedly the hepatotoxicity of the compound, as shown by measurements of the activities of appropriate hepatic enzymes in plasma.

Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals.

BACKGROUND: Large outbreaks of diarrhea caused by a newly recognized strain of Clostridium difficile occurred in four hospitals located in different parts of the United States between 1989 and 1992. Since frequent use of clindamycin was associated with the outbreak in one of the hospitals, we examined the resistance genes of the epidemic-strain isolates and studied the role of clindamycin use in these outbreaks. METHODS: Case-control studies were performed at three of the four hospitals to assess the relation of the use of clindamycin to C. difficile-associated diarrhea. All isolates of the epidemic strain and representative isolates of other strains identified during each outbreak were tested for susceptibility to clindamycin. Chromosomal DNA from these representative isolates was also analyzed by dot blot hybridization and amplification with the polymerase chain reaction (PCR) with the use of probes and primers from a previously described determinant of erythromycin resistance - the erythromycin ribosomal methylase B (ermB) gene - found in C. perfringens and C. difficile. RESULTS: In a stratified analysis of the case-control studies with pooling of the results according to the Mantel-Haenszel method, we found that the use of clindamycin was significantly increased among patients with diarrhea due to the epidemic strain of C. difficile, as compared with patients whose diarrhea was due to nonepidemic strains (pooled odds ratio, 4.35; 95 percent confidence interval, 2.02 to 9.38; P<0.001). Exposure to other types of antibiotics or hospitalization in a surgical ward was not significantly associated with the risk of C. difficile-associated diarrhea due to the epidemic strain. All epidemic-strain isolates were highly resistant to clindamycin (minimal inhibitory concentration, >256 microg per milliliter). DNA hybridization and PCR analysis showed that all these isolates had an ermB gene, which encodes a 23S ribosomal RNA methylase that mediates resistance to macrolide, lincosamide, and streptogramin antibiotics. Only 15 percent of the nonepidemic strains were resistant to clindamycin. CONCLUSIONS: A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.