Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction.

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, K(v)1.3, and [(3)H]dihydrocorreolide (diTC) binds with high affinity (K(d) approximately 10 nM) to membranes from Chinese hamster ovary cells that express K(v)1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F. , Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, K(v)3.2. Transferring the pore from K(v)1.3 to K(v)3.2 produces a chimera that binds peptidyl inhibitors of K(v)1.3 with high affinity, but not diTC. Transfer of the S(5) region of K(v)1.3 to K(v)3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with K(v)1.3, whereas transfer of the entire S(5)-S(6) domain results in a normal K(v)1.3 phenotype. Substitutions in S(5)-S(6) of K(v)1.3 with nonconserved residues from K(v)3.2 has identified two positions in S(5) and one in S(6) that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to K(v)3.2 after substitution of these three residues with the corresponding amino acids found in K(v)1.3. These results suggest that lack of sensitivity of K(v)3.2 to diTC is a consequence of the presence of Phe(382) and Ile(387) in S(5), and Met(458) in S(6). Inspection of K(v)1.1-1.6 channels indicates that they all possess identical S(5) and S(6) domains. As expected, diTC binds with high affinity (K(d) values 7-21 nM) to each of these homotetrameric channels. However, the kinetics of binding are fastest with K(v)1.3 and K(v)1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify K(v)1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.     

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.     

Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H₂O₂). The results indicated that salidroside exerted cytoprotective effects in an H₂O₂-induced HUVEC injury model and suppressed H₂O₂-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Motility and segregation of Hsp104-associated protein aggregates in budding yeast

During yeast cell division, aggregates of damaged proteins are segregated asymmetrically between the bud and the mother. It is thought that protein aggregates are cleared from the bud via actin cable-based retrograde transport toward the mother and that Bni1p formin regulates this transport. Here, we examined the dynamics of Hsp104-associated protein aggregates by video microscopy, particle tracking, and image correlation analysis. We show that protein aggregates undergo random walk without directional bias. Clearance of heat-induced aggregates from the bud does not depend on formin proteins but occurs mostly through dissolution via Hsp104p chaperon. Aggregates formed naturally in aged cells also exhibit random walk but do not dissolve during observation. Although our data do not disagree with a role for actin or cell polarity in aggregate segregation, modeling suggests that their asymmetric inheritance can be a predictable outcome of aggregates' slow diffusion and the geometry of yeast cells.     

Quantitation and significance of 125I-calmodulin binding to myosin light chain kinase and phosphorylase distributed on polyacrylamide gels

Glycogen phosphorylase (a or b) binds 125I-calmodulin in a Ca2+-dependent manner, in the 125I-calmodulin overlay technique. This binding is quantitatively identical to 125I-calmodulin binding to myosin light chain kinase. In an in vitro assay, calmodulin stimulates phosphorylase activity at limiting concentrations of either glucose-1-phosphate or glycogen, but the Ka is 1000 fold higher than for the kinase, and is not Ca2+-dependent. Activation of phosphorylase, but not myosin light chain kinase, by calmodulin can be mimicked by troponin C or bovine serum albumin. These results demonstrate that the properties of calmodulin interaction with proteins can vary between the 125I-calmodulin technique and a functional assay of calmodulin effect on the same protein.