Influence of voltage and extracellular Na+ on amiloride block and transport kinetics of rat epithelial Na+ channel expressed in Xenopus oocytes

We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.     

Utility of immunohistochemistry in detecting alterations of mismatch repair genes of DNA. A series of 48 cases of colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) is associated in more than 95% to a germline mutation in the genes of the mismatch repair (MMR) of DNA. The aim of this study was to assess the utility of immunohistochemistry, a simple and fast technique, in the triage of families where HNPCC is suspected. Tumor samples included in this study were from patients with resection for colorectal cancer, examined in our laboratory between 2004 and 2007. For each case, a formalin-fixed paraffin-embedded tissue block containing tumor tissue and normal adjacent mucosa was selected. Tumor specimens were examined with immunohistochemistry for the presence of hMLH1, hMSH2, and hMSH6 proteins. Scoring of the tumor staining was performed without any knowledge of patients' family history. The loss of protein expression was noted in four patients among 48?cases tested: two cases with isolated loss of hMSH2, a case with isolated loss of hMSH6 and one case with combined loss of MSH2/MSH6. No case has shown a suppression of hMLH1 protein. Comparing the immunohistochemical results for clinical has revealed a clear correlation between loss of protein expression demonstrated by immunohistochemistry and clinical data. Indeed, three cases among the four who showed no expression of MMR proteins showed at least one clinical criterion predictive of HNPCC. In conclusion, our study support the potential utility of immunohistochemistry to identify a significant portion of colorectal tumors derived from germline mutation of MMR genes and can be used as an adjunct measure in the identification of HNPCC.     

Analysis of FMR1 gene premutation and X chromosome cytogenetic abnormalities in 100 Tunisian patients presenting premature ovarian failure

Objective

To evaluate the prevalence of FMR1 premutations and X chromosome cytogenetic abnormalities in a large cohort of Tunisian women with premature ovarian failure (POF).

Patients and methods

The cohort consisted of 127 Tunisian women with POF referred by endocrinologists and gynecologists for genetic investigation in the context of idiopathic POF and altered hormonal profiles. Clinical information concerning the reproductive function in the family, previous hormonal measurements and/or possible fertility treatment were collected. Karyotype, FISH analyses, FMR1 and FMR2 testing were performed for all patients.

Results

Fifteen patients (11.81%) presented structural or numerical X chromosomal abnormalities. Moreover, we detected in 12 patients (10.71%) a high level of X mosaicism. Analysis of FMR1 gene in the 100 patients without X chromosomal abnormalities showed that five percent of the patients carried a FMR1 premutation allele. On the other hand, the FMR2 screening did not reveal any deletion.

Conclusion

Our study confirms the major role of X chromosome abnormalities in POF and highlights the importance of karyotype analyses and FMR1 screening. These investigations provide valuable information for diagnosis and genetic counseling for these women who still have a 5% chance of spontaneous conception.     

Place of the precocious surgical treatment in facial hemangioma

Infantile cutaneous hemangioma is a benign vascular tumour present at 10% of the infants. It forms part of the group of the vascular tumours in the classification of international society for vascular anomalies (ISSVA). Usual surgical attitude is abstention and surgery is proposed only in order to treat sequelae. But some particular situations require early surgery to avoid functional impairment, deformation or growth delay due to the lesion's development. Using our observations, we recall the epidemiology, the physiopathology, the clinical aspects, the particularities of the facial localizations and their treatments. In these localizations the time intervenes like a fourth dimension that is going to modify, to improve or to aggravate the prognosis. Treatment requires a strategy and precocious surgery. We insist on the fact that the dogma of the therapeutic abstention remained true for a majority of children with small size hemangioma and that a precocious surgery must be proposed for some localizations in the face.     

Acceptability of coupling intermittent preventive treatment in infants with the expanded programme on immunization in three francophone countries in Africa

Objective Intermittent preventive treatment in infants (IPTi) is a malaria control strategy currently recommended by WHO for implementation at scale in Africa, consisting of administration of sulphadoxine‐pyrimethamine (SP) coupled with routine immunizations offered to children under 1 year. In this study, we analysed IPTi acceptability by communities and health staff. Methods Direct observation, in‐depth interviews (IDIs) and focus group discussions (FGDs) were conducted in Benin, Madagascar and Senegal during IPTi pilot implementation. Villages were stratified by immunization coverage. Data were transcribed and analysed using NVivo7 software. Results Communities’ knowledge of malaria aetiology and diagnosis was good, although generally villagers did not seek treatment at health centres as their first choice. Perceptions and attitudes towards IPTi were very positive among communities and health workers. A misconception that SP was an antipyretic that prevents post‐vaccinal fever contributed to IPTi’s acceptability. No refusals or negative rumours related to IPTi coupling with immunizations were identified, and IPTi did not negatively influence attitudes towards other malaria control strategies. Healthcare decisions about children, normatively made by the father, are starting to shift to educated and financially independent mothers. Discussion Intermittent preventive treatment in infants is well accepted by providers and communities, showing a synergic acceptability when coupled with routine immunizations. However, a misconception that SP alleviates fever should be addressed when scaling up implementation.