Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)     

Effects of hyperthyroidism on delayed rectifier K+ currents in left and right murine atria

Hyperthyroidism has been associated with atrial fibrillation (AF); however, hyperthyroidism-induced ion channel changes that may predispose to AF have not been fully elucidated. To understand the electrophysiological changes that occur in left and right atria with hyperthyroidism, the patch-clamp technique was used to compare action potential duration (APD) and whole cell currents in myocytes from left and right atria from both control and hyperthyroid mice. Additionally, RNase protection assays and immunoblotting were performed to evaluate the mRNA and protein expression levels of K(+) channel alpha-subunits in left and right atria. The results showed that 1) in control mice, the APD was shorter and the ultra-rapid delayed rectifier K(+) conductance (I(Kur)) and the sustained delayed rectifier K(+) conductance (I(ss)) were larger in the left than in the right atrium; also, mRNA and protein expression levels of Kv1.5 and Kv2.1 were higher in the left atrium; 2) in hyperthyroid mice, the APD was shortened and I(Kur) and I(ss) were increased in both left and right atrial myocytes, and the protein expression levels of Kv1.5 and Kv2.1 were increased significantly in both atria; and 3) the influence of hyperthyroidism on APD and delayed rectifier K(+) currents was more prominent in right than in left atrium, which minimized the interatrial APD difference. In conclusion, hyperthyroidism resulted in more significant APD shortening and greater delayed rectifier K(+) current increases in the right vs. the left atrium, which can contribute to the propensity for atrial arrhythmia in hyperthyroid heart.     

International Union of Physiological Sciences teaching workshop, April 7-10, 2005, Pali Mountain, California

Fifty-six physiologists from fourteen countries participated in the sixth International Union of Physiological Sciences Teaching Workshop. The four-day program included a poster session, a debate on integrative versus disciplinary curricula, and interactive lectures on evidence-based education, inquiry laboratories, and student preparation for physiology courses. Participants worked in small groups on one of four topics: developing free web-based laboratory resources, information technology, curriculum planning and design, and other issues in classroom teaching.     

Nuclear fusion occurs during mating in Candida albicans and is dependent on the KAR3 gene

It is now well established that mating can occur between diploid a and alpha cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/alpha cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.     

Inactivation of endotoxin by human plasma gelsolin

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.     

Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration

It has been generally accepted that enzyme activity requires a minimal hydration of about 0.2 g H2O g(-1) protein. This fits well with evidence that hydration above this level is associated with the onset of intramolecular motions. The influence of enzyme hydration on the hydrolysis of substrate by Candida rugosa Lipase B and pig liver esterase was investigated. Each enzyme was studied as a powder at various hydration levels, using vapour phase ethyl butyrate as substrate. This procedure allows the separation of those effects that are due to hydration from those arising from diffusional constraints. We found hydrolytic activity in both enzymes at all hydration levels above zero (between 0.054-0.47 and 0.029-0.60 g H2O g(-1) protein, respectively) that were investigated. The lowest hydration level investigated, <0.03 g H2O g(-1) enzyme, corresponded to a water/enzyme mole ratio of 100 and a coverage of about 10% of the enzyme surface by water molecules. The hydrolytic activity of both enzymes was dependent on protein hydration. However, since the hydrolysis of ethyl butyrate requires water as a second substrate, the absence of activity at zero hydration does not rule out the possibility of enzyme activity in the absence of water. These results suggest that the properties conferred on proteins by water, at least above 10% surface coverage (in this case corresponding to a hydration level of 0.03 g H2O g(-1) protein), are not a requirement for enzyme catalysis.     

The human papillomavirus 16 E6 protein binds to Fas-associated death domain and protects cells from Fas-triggered apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of the tumor suppressor p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that E6 can protect cells from tumor necrosis factor-induced apoptosis by binding to the C-terminal end of tumor necrosis factor R1, thus blocking apoptotic signal transduction. In this study, E6 was shown to also protect cells from apoptosis induced via the Fas pathway. Furthermore, use of an inducible E6 expression system demonstrated that this protection is dose-dependent, with higher levels of E6 leading to greater protection. Although E6 suppresses activation of both caspase 3 and caspase 8, it does not affect apoptotic signaling through the mitochondrial pathway. Mammalian two-hybrid and in vitro pull-down assays were then used to demonstrate that E6 binds directly to the death effector domain of Fas-associated death domain (FADD), with deletion and site-directed mutants enabling the localization of the E6-binding site to the N-terminal end of the FADD death effector domain. E6 is produced in two forms as follows: a full-length version of approximately 16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6, was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway.     

How small peptides block and reverse serpin polymerisation

Many of the late-onset dementias, including Alzheimer's disease and the prion encephalopathies, arise from the aberrant aggregation of individual proteins. The serpin family of serine protease inhibitors provides a well-defined structural example of such pathological aggregation, as its mutant variants readily form long-chain polymers, resulting in diseases ranging from thrombosis to dementia. The intermolecular linkages result from the insertion of the reactive site loop of one serpin molecule into the middle strand (s4A) position of the A beta-sheet of another molecule. We define here the structural requirements for small peptides to competitively bind to and block the s4A position to prevent this intermolecular linkage and polymerisation. The entry and anchoring of blocking-peptides is facilitated by the presence of a threonine which inserts into the site equivalent to P8 of s4A. But the critical requirement for small blocking-peptides is demonstrated in crystallographic structures of the complexes formed with selected tri- and tetrapeptides. These structures indicate that the binding is primarily due to the insertion of peptide hydrophobic side-chains into the P4 and P6 sites of s4A. The findings allow the rational design of synthetic blocking-peptides small enough to be suitable for mimetic design. This is demonstrated here with a tetrapeptide that preferentially blocks the polymerisation of a pathologically unstable serpin commonly present in people of European descent.     

Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.