Rpa12p, a conserved RNA polymerase I subunit with two functional domains

Rpa12p is a subunit of RNA polymerase I formed of two zinc-binding domains. The N-terminal zinc region (positions 1-60) is poorly conserved from yeast to man. The C-terminal domain contains an invariant Q.RSADE.T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N-terminal domain fail to grow at 34 degrees C, are sensitive to nucleotide-depleting drugs and become lethal in rpa14-Delta mutants lacking the non-essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two-hybrid interaction with the second largest subunit. In all these respects, N-terminal deletions behave like full deletions. In contrast, C-terminal deletions retaining only the first N-terminal 60 amino acids are indistinguishable from wild type. Thus, the N-terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.     

Proteinase-activated receptor-2-induced colonic inflammation in mice: possible involvement of afferent neurons,nitric oxide,and paracellular permeability

Activation of colonic proteinase-activated receptor-2 (PAR-2) provokes colonic inflammation and increases mucosal permeability in mice. The mechanism of inflammation is under debate and could be neurogenic and/or the consequence of tight-junction opening with passage of exogenous pathogens into the lamina propria. The present study aimed to further characterize the inflammatory effect of PAR-2 activation by investigating: 1) the role of NO, 2) the role of afferent neurons, and 3) a possible cause and effect relationship between colonic paracellular permeability changes and mucosal inflammation. Thus, intracolonic infusion to mice of the PAR-2-activating peptide, SLIGRL, increased both myeloperoxidase (MPO) activity and damage scores indicating colonic inflammation, and enhanced colonic permeability to (51)Cr-EDTA from 2 to 4 h after its infusion. NO synthase inhibitors, L-NAME and aminoguanidine, as well as the neurotoxin capsaicin and NK1, calcitonin gene-related peptide (CGRP) receptor antagonists, SR140333 and CGRP(8-37), prevented SLIGRL-induced MPO and damage score increases and permeability. In contrast, although the tight-junction blocker, 2,4,6-triaminopyrimidine, and the myosin L chain kinase inhibitor, ML-7, prevented SLIGRL-induced increase in permeability, they did not prevent MPO and damage score increases. Taken together our data show that both NO and capsaicin-sensitive afferent neurons are involved in PAR-2-mediated colonic inflammation and paracellular permeability increase. Nevertheless, the inflammation process is not a consequence of increased permeability which results at least in part from the activation of myosin L chain kinase.     

Proliferative lifespan is conserved after nuclear transfer

Cultured primary cells exhibit a finite proliferative lifespan, termed the Hayflick limit. Cloning by nuclear transfer can reverse this cellular ageing process and can be accomplished with cultured cells nearing senescence. Here we describe nuclear transfer experiments in which donor cell lines at different ages and with different proliferative capacities were used to clone foetuses and animals from which new primary cell lines were generated. The rederived lines had the same proliferative capacity and rate of telomere shortening as the donor cell lines, suggesting that these are innate, genetically determined, properties that are conserved by nuclear transfer.     

Increased expression of the gene for alpha-interferon-inducible protein in cardiomyopathic hamster heart

Cardiomyopathic (CM) hamsters have a disruption in the delta-sarcoglycan gene which leads to progressive cardiac necrosis by 30 to 40 days of age, hypertrophy by 120 days, and heart failure by 250 days. We used differential display to detect other changes in mRNA levels in 30-, 60-, and 90-day-old wild-type and CM hamsters. We identified a 400-bp cDNA with sequence similarity to the human alpha-interferon-inducible protein (p27). This cDNA annealed with a 570-base mRNA whose steady-state levels were increased in 30-, 60-, and 90-day-old CM compared to wild-type heart. Increased expression of this hamster homolog of p27 (p27-h) was detected in CM hamster cardiac and skeletal muscle at 60 days of age but not in liver, kidney, or brain. Thus, an inherited defect in CM hamsters leads to increased expression of p27-h in advance of the development of hypertrophy and heart failure.     

Chronological approach of diet-induced alterations in muscle mitochondrial functions in rats

Objective: Mitochondrial dysfunction might predispose individuals to develop insulin resistance. Our objective was to determine whether mitochondrial dysfunction or insulin resistance was the primary event during high‐fat (HF) diet. Research Methods and Procedures: Rats were fed an HF diet for 0, 3, 6, 9, 14, 20, or 40 days and compared with control. Soleus and tibialis muscle mitochondrial activity were assessed using permeabilized fiber technique. Insulin [area under the curve for insulin (AUC_I)] and glucose [area under the curve for glucose (AUC_G)] responses to intraperitoneal glucose tolerance test as well as fasting plasma non‐esterified fatty acids (NEFAs), triglyceride, and glycerol concentrations were determined. Results: AUC_I and AUC_G were altered from Day 6 (p < 0.01 vs. Day 0). In soleus, oxidative phosphorylation (OXPHOS) activity was transiently enhanced by 26% after 14 days of HF diet (p < 0.05 vs. Day 0) conjointly with 62% increase in NEFA concentration (p < 0.05 vs. Day 0). This was associated with normalized AUC_G at Day 14 and with a decline of plasma NEFA concentration together with stabilization of intra‐abdominal adiposity at Day 20. Prolongation of HF diet again caused an increase in plasma NEFA concentration, intra‐abdominal adiposity, AUC_I, and AUC_G. At Day 40, significant decrease in OXPHOS activity was observed in soleus. Discussion: Mitochondria first adapt to overfeeding in oxidative muscle limiting excess fat deposition. This potentially contributes to maintain glucose homeostasis. Persistent overfeeding causes insulin resistance and results in a slow decline in oxidative muscle OXPHOS activity. This shows that the involvement of mitochondria in the predisposition to insulin resistance is mainly due to an inability to face prolonged excess fat delivery.     

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

Background

Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K⁺ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before.

Results

Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair.

Conclusion

Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.