Inhibition of connexin 43 in cardiac muscle during intense physical exercise.

Endurance training is accompanied by important adaptations in both cardiovascular and autonomic nervous systems. Previous works have shown that the main component of gap junctions in the ventricular myocardium (connexin 43 (Cx43) can be regulated by adrenergic stimulus. On the other hand, training raises vagal and decreases sympathetic tone, while augmenting myocardial sensitivity to sympathetic stimulation during exercise. We therefore evaluated the regulation of Cx43 expression by sympathetic tone during exercise in trained and sedentary mice. Training induced an increase in the protein level of Cx43 by 45-70% under resting conditions. The expression of Cx43 was inhibited in trained but not in untrained mice in response to a 60 min exercise bout. Normal basal expression was restored after 60 min of resting. Cx43 reached a minimum that was not different from basal expression in untrained mice. In accordance, electrocardiography and action potential analysis did not reveal major electrophysiological implications for the drop in Cx43 abundance in trained-exercise mice. We prevented Cx43 inhibition using propranolol, and observed increased basal mRNA levels of ß-adrenergic receptors without significant changes in the ratio ß1 to ß2. In conclusion, we showed that Cx43 expression is transiently inhibited by ß-adrenergic stimulus in trained mice during acute exercise.

Low Vitamin D Levels at Birth and Early Respiratory Outcome in Infants With Gestational Age Less Than 29 Weeks.

BACKGROUND: Vitamin D (VitD) is involved in lung development but its influence on respiratory distress syndrome of extremely preterm (EPT) infants have been little investigated. In this study, we examined the influence of low vitamin D status at birth on early respiratory outcomes of this vulnerable infant population. METHODS: Cord blood 25(OH)D levels ≤ 75 nmol/L were considered as Low vitamin D levels. Stepwise logistic regression and classification regression-tree analyses were used and the primary outcome was the combined outcome of death or mechanical ventilation need by the end of the first week (death or MV DoL7) as a marker od RDS severity. RESULTS: The mean (SD) GA and birth weight were 26 (1.4) weeks and 801 (212) gr, respectively; 81/109 (74%) infants had low 25(OH)D levels. Infants with low VitD levels had 25% higher initial FiO2 levels (p < 0.05) and were more likely to be mechanically ventilated on DoL7 (36 vs. 7%, p < 0.05). Adjusted for gestational age, they had 10-fold higher odds of death or MV DoL7 (p < 0.01). By regression tree analysis, the rate of death or MV DoL7 increased from 18 to 71% in infants with GA < 26 weeks and with cord blood 25(OH)D levels higher and lower than 74 nmol/L, respectively (p < 0.05). CONCLUSION: Low vitamin D levels at birth are associated with early adverse respiratory outcomes in infants with GA less 29 weeks. Further largest studies are needed to confirm this association.

Expression and activity of a recombinant chimeric protein composed of pokeweed antiviral protein and of human interleukin-2.

The pokeweed antiviral protein (PAP) has already been used to chemically construct immunotoxins. Here we tested the recombinant approach for the production of PAP-containing cytotoxic fusion-proteins. A cDNA encoding a mutated PAP (PAP9), which is expressed at high levels in bacteria, was fused to human interleukin-2 (IL-2) cDNA. The resulting PAP9-IL-2 protein was as active as the free PAP9 in inhibiting an eukaryotic cell-free translation system. Only the chimeric protein desaminated the 28S rRNA and inhibited translation of the CTLL-2 cell line which expresses the IL-2 receptor. These results show that PAP is a suitable toxin for the production of recombinant immunotoxins.

Application of microsatellite PCR techniques in the identification of mixed up tissue specimens in surgical pathology.

A fragment of tumour was erroneously mixed up with an endometrial biopsy. Micro-satellite polymerase chain reaction (PCR) clearly demonstrated the extraneous nature of the fragment. Micro-satellite PCR may be useful for the identification of mis-labelled or mismatched tissue fragments in surgical pathology specimens.

[A reproducibility and validity study of a systematic review on ischemic cardiopathy. The Study Group of the Quality of Care (GRECA)]. / Estudio de la reproducibilidad y validez de una revisión sistemática sobre cardiopatía isquémica. Grupo de Estudio de la Calidad Asistencial (GRECA).

BACKGROUND: The systematic literature reviews have been proposed as a method of scientific evidence identification since they protect the final product from the subjectivity of each primary source reviewer. However, it is not known whether the different ways of evidence synthesis accomplish suitable criteria of objectivity, reliability and biases protection so as to be considered scientifically valid. An experiment of reliability and validity of a systematic literature review about coronary heart disease was carried out. MATERIAL AND METHODS: Study of blind concordance between two independent reviewers for the identification, selection, retrieval and quality evaluation of the articles by using the same protocol. The concordance was analysed by the kappa index for two observers in different categories. The validity was evaluated throughout the acceptability of the review users. RESULTS: The concordance for their identification capacity was poor although they used the same key words (869 versus 476). But the concordance improved when considering selection (26.6% versus 29.2%), retrieval (agreement = 76%) classification by kind of article (kappa = 0.60) and scoring by strength of the evidence (kappa = 0.87). The acceptability was high among review users. CONCLUSIONS: It would be assumed that, even under tight rules of performance, the systematic literature reviews are not completely protected against some biases which could damage their validity in a non easily controllable form. The implication of reviewers, experts in documentation and users of the literature, together with pilot studies performed previous to the review, seems to be the best way to yield better results.

Mutations dissociating the inhibitory activity of the pokeweed antiviral protein on eukaryote translation and Escherichia coli growth.

The pokeweed antiviral protein is a ribosome inactivating protein acting on eukaryotic as well as on prokaryotic ribosomes thus is toxic for both cell types. Using the PCR technique to clone the PAP open reading frame, we characterized two cDNAs coding for proteins inhibiting eukaryotic translation process and which are not toxic for Escherichia coli, unlike the wild type protein. The sequence of the two cDNAs showed that the proteins contain only one and two point mutations. This result suggest that the wild type amino acids in the mutated positions participate in the prokaryotic ribosome recognition. These mutants might be useful for the construction of immunotoxins containing the pokeweed antiviral protein as toxin.

Potent acetylcholinesterase inhibitors: design, synthesis and structure-activity relationships of alkylene linked bis-galanthamine and galanthamine-galanthaminium salts.

The syntheses, the anticholinesterase activities and structure-activity relationships of homodimeric (3a-c) and heterodimeric (6a-c) alkylene linked bis-galanthamine are reported. Compounds 6b-c were found to be more potent than galanthamine and tacrine in inhibiting AChE.

Loss of heterozygosity at the RB-1 locus and pRB immunostaining in epithelial ovarian tumors: a molecular, immunohistochemical, and clinicopathologic study.

Alterations in the retinoblastoma gene (RB-1) are common in human neoplasia. The frequency of loss of heterozygosity (LOH) at the RB-1 locus on chromosome 13q14 was studied in a series of 51 epithelial ovarian tumors (10 benign, 7 borderline, and 34 malignant). LOH was scored by the absence or reduction of the signal to < 50% of one of the alleles in tumor DNA compared with normal DNA. LOH results were correlated with retinoblastoma protein (pRB) immunostaining. LOH at the RB-1 locus was observed in 9 tumors (17.6%), specifically in 1 of 7 borderline tumors and 8 of 34 ovarian carcinomas (23.5%). Among the malignant tumors, LOH occurred more frequently in carcinomas with serous differentiation (7/23; 30%). A heterogeneous (10% to 70% cells) or diffuse (> 70% cells) pRB immunostaining was less frequent in benign (1/10; 10%) and borderline (2/7; 28%) tumors than in ovarian carcinomas (15/34; 44%), an observation that correlated with the higher proliferative index in carcinomas than in benign and borderline tumors. However, lack or only focal (< 10% cells) pRB immunostaining occurred in the vast majority of tumors with LOH at the RB-1 locus (7/9; 77%), a finding that may suggest a tumor suppressor role for RB-1 in these tumors. The results suggest that RB-1 may play a role in a subset of ovarian carcinomas, particularly those exhibiting serous differentiation.

Glucocorticoid-mediated destabilization of cyclin D3 mRNA involves RNA-protein interactions in the 3'-untranslated region of the mRNA.

Glucocorticoids regulate the expression of the G(1) progression factor, cyclin D3. Cyclin D3 messenger RNA (CcnD3 mRNA) stability decreases rapidly when murine T lymphoma cells are treated with the synthetic glucocorticoid dexamethasone. Basal stability of CcnD3 mRNA is regulated by sequences within the 3'-untranslated region (3'-UTR). RNA-protein interactions occurring within the CcnD3 3'-UTR have been analyzed by RNA electrophoretic mobility shift assay. Three sites of RNA-protein interaction have been mapped using this approach. These elements include three pyrimidine-rich domains of 25, 26, and 37 nucleotides. When the cyclin D3 3'-UTR was stably overexpressed, the endogenous CcnD3 mRNA was no longer regulated by dexamethasone. Likewise, overexpression of a 215-nucleotide transgene that contains the 26- and 37-nucleotide elements blocks glucocorticoid inhibition of CcnD3 mRNA expression. These observations suggest that the 215-nucleotide 3'-UTR element may act as a molecular decoy, competing for proteins that bind to the endogenous transcript and thereby attenuating glucocorticoid responsiveness. UV-cross-linking experiments showed that two proteins of approximate molecular weight 37,000 and 52,000 bind to this 3'-UTR element.

Loss of heterozygosity on chromosome 13q12-q14, BRCA-2 mutations and lack of BRCA-2 promoter hypermethylation in sporadic epithelial ovarian tumors.

BACKGROUND: BRCA-1 and BRCA-2 are tumor suppressor genes in familial breast-ovarian carcinoma syndrome. BRCA-1 is also a tumor suppressor gene in sporadic ovarian carcinomas. However, the role of BRCA-2 in sporadic ovarian tumors remains unclear. METHODS: DNA from 52 patients with clinically apparent sporadic ovarian tumors was extracted from blood and from fresh-frozen tumor tissue and normal tissue (10 benign, 7 borderline, and 35 malignant). Loss of heterozygosity (LOH) was analyzed in six microsatellite loci on chromosome 13q. BRCA-2 mutations were detected by single-strand conformation polymorphism analysis and the protein truncation test. BRCA-2 promoter methylation was evaluated by methylation specific polymerase chain reaction analysis. RESULTS: LOH on chromosome 13q12-q14 was identified in 16 tumors (30.8%): Fifteen of these tumors were carcinomas (15 of 35 tumors; 42.8%) and one was a borderline tumor. LOH was frequent in carcinomas with serous differentiation (12 of 16 tumors; 75%). LOH on chromosome 13q12-q14 coexisted with LOH on chromosome 17q in 10 carcinomas. BRCA-2 methylation was not detected in any tumor. BRCA-2 mutations were found in three tumors (one somatic nonsense and two germline frameshift). BRCA-2 fulfilled the two hits for a tumor suppressor gene in these three tumors; in one of them, a BRCA-1 tumor suppressor role had been demonstrated previously. CONCLUSIONS: The results suggest that BRCA-1 and BRCA-2 may act synergically in sporadic ovarian carcinomas with serous differentiation. The demonstration of BRCA-2 germline mutations in patients with ovarian carcinoma with LOH on chromosome 13q12-q14 and lack of a remarkable family history of cancer suggest that the proportion of ovarian carcinomas that result from hereditary predisposition may be higher than previously estimated.

Microsatellite instability, MLH-1 promoter hypermethylation, and frameshift mutations at coding mononucleotide repeat microsatellites in ovarian tumors.

BACKGROUND: Microsatellite instability (MI) is frequent in endometrial carcinomas (ECs), but its occurrence in ovarian tumors is uncertain. Microsatellite instability positive ECs frequently are associated with frameshift mutations in coding mononucleotide tracts in IGFIIR, BAX, hMSH6, and hMSH3. METHODS: DNA from 52 consecutive patients with ovarian tumors (10 benign, 7 borderline, and 35 malignant) was obtained from neoplastic and normal tissue. After preliminary results, the series was expanded by including 41 additional, previously selected, endometrioid and clear cell carcinomas. Microsatellite instability analysis was assessed by evaluating three (CA)n dinucleotide repeats (D2S123, D5S346, D17S250) and two mononucleotide tracts (BAT 25 and BAT 26). Frameshift mutations at coding mononucleotide repeats (IGFIIR, TGF beta II, BAX, hMSH6, and hMSH3) were investigated by single-strand conformation polymorphism analysis and DNA sequencing. MLH-1 methylation was assessed by methylation specific PCR. RESULTS: Microsatellite instability was identified in only 2 of the 52 (3.8%) tumors of the initial series (1 endometrioid and 1 clear cell carcinoma). After expanding the initial series of 15 endometrioid and clear cell carcinomas with 41 additional endometrioid and clear cell carcinomas, MI was found in 7 of the total series of 56 endometrioid and clear cell carcinomas (12.5%). Frameshift mutations in coding mononucleotide tracts were detected in BAX (6 of 7), IGFIIR (1 of 7), and MSH3 (2 of 7). MLH-1 promoter hypermethylation was identified in three of six MI positive tumors. CONCLUSIONS: Microsatellite instability was infrequent in this series of ovarian tumors, and it was limited to endometrioid and clear cell carcinomas. Like EC, many ovarian carcinomas with MI follow the same process of MLH-1 promoter methylation and accumulation of mutations in coding mononucleotide tracts.

PCR analysis in the pathological diagnosis of Whipple's disease: emphasis on extraintestinal involvement or atypical morphological features.

PCR analysis of species-specific bacterial 16S rRNA gene of Tropheryma whippelii was performed in biopsies from 10 cases of Whipple's disease (WD). In seven patients showing the typical clinical picture of WD, PCR was performed on the diagnostic intestinal biopsy. In the remaining three cases (an autopsy case of disseminated WD and two patients showing lymphadenopathy as the initial clinical presentation), PCR was done on lymph node specimens. In one of the lymph node biopsies, an unusual sarcoidlike granulomatous reaction had led to the diagnosis of sarcoidosis. The specific bacterial DNA was detected in all cases, both in intestinal biopsies and in lymph node specimens. Follow-up biopsies after antibiotic therapy were evaluated in two patients. The two follow-up biopsies were negative, although in both of them scattered nests of PAS-positive macrophages remained. The results of this study suggest that PCR analysis of species-specific sequences of the 16S rRNA of Tropheryma whippelii is a very useful tool for the pathological diagnosis of WD. It confirms the diagnosis of WD in intestinal biopsies as well as in extraintestinal sites, even when the morphological appearance is not typical. It is also the most precise technique for monitoring therapeutic effects.