Molekulare Charakterisierung des epithelialen Na+-Kanals (ENaC) aus Nasengewebe von Patienten mit und ohne Mukoviszidose

Mukoviszidose (Cystische Fibrose, CF) beruht auf einem Gendefekt im CFTR und ist charakterisiert durch eine reduzierte Cl-Sekretion und eine drastisch erhöhte Na+-Absorption, die durch ENaC hervorgerufen wird. Während viele CFTR-Mutationen untersucht und beschrieben wurden, blieben die molekularen Mechanismen, welche die Na+-Hyperabsorption durch ENaC verursachen, weitgehend unaufgeklärt. Um Mutationen im ENaC ausschließen zu können, die eine veränderte Kanaleigenschaft hervorrufen würden und somit zu einer gesteigerten Na+-Absorption führen könnten, wurde der humane ENaC aus respiratorischem Nasenepithel (hnENaC) von CF und nicht-CF Patienten kloniert und sequenziert. So konnte deutlich gezeigt werden, dass die Sequenzen des hnENaC von CF-Patienten und nicht-CF Patienten identisch sind. Nachgewiesen werden konnte darüber hinaus, dass die Menge an ENaC im Nasenepithel von CF Patienten auf mRNA- und teilweise auch Protein-Ebene im direkten Vergleich mit den nicht-CF Proben erhöht ist

Upregulated expression of ENaC in human CF nasal epithelium

AbstractCystic fibrosis (CF) is characterised by the absence of CFTR function resulting in a reduced Cl− secretion and an increase in Na+ absorption. This Na+ hyperabsorption is mediated by the human amiloride-sensitive epithelial sodium channel (ENaC), but the underlying mechanisms are still unknown. After demonstrating functional differences of the Na+ absorption in CF and non-CF epithelia in Ussing chamber experiments with human primary cultures, we compared ENaC sequences from CF and non-CF human nasal tissue (hnENaC), investigated the mRNA transcription levels via real-time PCR and studied the protein expression in Western blot analyses. We found no differences in the sequences of CF and non-CF hnENaC, but identified some polymorphisms. The real-time experiments revealed an enhanced mRNA amount of all three hnENaC subunits in CF tissue. By comparing the two groups on the protein level, we observed differences in the abundance of the Na+ channel. While the α- and β-hnENaC protein amount was increased in CF tissue the γ-hnENaC was decreased. We conclude that the Na+ hyperabsorption in CF is not caused by mutations in hnENaC, but by an increase in the transcription of the hnENaC subunits. This could be induced by a disturbed regulation of the channel in CF

Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria.

Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics

Feasibility of needle and syringe programs in Tajikistan distributing low dead space needles

Background In 2012, the World Health Organization recommended that needle and syringe programs offer their clients low dead space insulin syringes with permanently attached needles. However, in many countries, these syringes are not acceptable to a majority of people who inject drugs. This study assessed the feasibility of working with needle and syringe programs to implement the WHO recommendation using low dead space detachable needles. The study also assessed the acceptability of the needles. Methods Two needle and syringe programs in Tajikistan—one in Kulob and one in Khudjand—received 25,000 low dead space detachable needles each. The programs distributed low dead space detachable needles and a marketing flyer that emphasized the relative advantages of the needles. Each program also enrolled 100 participants, and each participant completed a baseline interview and a 2-month follow-up interview. Results At follow-up, 100% of participants reported trying the low dead space detachable needles, and 96% reported that they liked using the needles. Both needle and syringe programs distributed all their needles within the first 60 days of the project indicating use of the needles, even among clients who did not participate in the study. Conclusions This project demonstrates that it is feasible for needle and syringe programs to offer and promote low dead space needles to their clients. The findings indicate that low dead space needles are acceptable to needle and syringe program clients in these Tajikistan cities. To reduce HIV and hepatitis C virus transmission, needle and syringe programs should offer low dead space needles, low dead space insulin syringes in addition to standard needles, and syringes to their clients