A fundamental role for KChIPs in determining the molecular properties and trafficking of Kv4.2 potassium channels

Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 alpha subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 alpha subunits. Co-expression of KChIPs1-3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.     

Architecture of the budding yeast kinetochore reveals a conserved molecular core

How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.     

Quantitative isolation of microbial DNA from the different types of soils of natural and agricultural ecosystems

A novel procedure was developed for direct quantitative isolation of microbial DNA from soil. This technique was used to evaluate microbial DNA pools in soils of contrasting types (chernozems and brown forest soils) under different anthropogenic loads. A strong correlation was found between microbial biomass and DNA contents in soils of different types (R2 = 0.799). The ratio of soil CO2 emission rate to the amount of extractable DNA in the soil was shown to reflect physiological state of the soil microbial community; this ratio can be used as an ecophysiological parameter similarly to the metabolic quotient qCO2.     

Systematics of Halosarpheia based on morphological and molecular data

The genus Halosarpheia (Halosphaeriales) was established for marine ascomycetes with obpyriform to sub-globose, coriaceous, brown to black ostiolate ascomata with long necks; hamathecia of catenophyses; thin-walled, unitunicate, persistent asci with thick-walled apices; and ellipsoid, one septate, hyaline ascospores equipped with coiled, threadlike apical appendages that unfurl in water. Emphasis on ascospore appendage morphology has led to the inclusion in the genus of morphologically disparate fungi from a variety of marine and freshwater habitats. To better understand the evolutionary relationships of Halosarpheia species, phylogenetic analyses were conducted on 16 Halosarpheia species, 13 other species of Halosphaeriales and representatives of the Microascales, Hypocreales, Sordariales and Xylariales using 18S and 28S rDNA sequence data. All of the Halosarpheia species occurred on the Halosphaeriales clade. The type species of the genus, H. fibrosa, occurred on a well-supported clade with two morphologically similar species, H. trullifera and H. unicellularis. This clade, which phylogenetically was distant from the clades of other Halosarpheia species, represents the genus Halosarpheia sensu stricto. The other Halosarpheia species were distributed among eight other well-supported clades clearly separated from one another based on molecular data. New generic names are established for six of these clades, one new species is described, and one species is transferred to Aniptodera. A table (Table I) comparing the morphology, habitat, substrate and distribution of the genera of aquatic ascomycetes with coiled, threadlike apical appendages treated in this study is provided, along with a key for their identification.     

Confirmation of linkage of prostate cancer aggressiveness with chromosome 19q

Regions on chromosomes 7 and 19 were recently reported to contain susceptibility loci that regulate tumor aggressiveness of prostate cancer. To confirm these findings, we analyzed genome scan data from 161 pedigrees affected with prostate cancer. Using the Gleason score as a quantitative measure of tumor aggressiveness, we regressed the squared trait difference, as well as the mean-corrected cross product, on the estimated proportion of alleles shared identical-by-descent at each marker position. Our results confirm the previous linkage results for chromosome 19q (D19S902, P<.00001). In addition, we report suggestive evidence for linkage on chromosome 4 (D4S403, P=.00012). The results of previous findings, together with our results, provide strong evidence that chromosome 19 harbors a gene for tumor aggressiveness.     

The synergistic activation of Raf-1 kinase by phorbol myristate acetate and hydrogen peroxide in NIH3T3 cells

We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-epsilon activation as determined by the transient expression of dominant negative mutants of PKC-epsilon-K436R. Interestingly, H(2)O(2) produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H(2)O(2). This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H(2)O(2) occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-epsilon, along with a transient phosphorylation of both Raf-1 kinase and PKC.     

Isolation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils

Cathelicidins are a family of gene-encoded antimicrobial peptides found in mammals. Seven cathelicidin genes have been identified in sheep, but up to now only two variants of one of these predicted peptides (OaBac5) have been purified from ovine neutrophils. In this work numerous proline/arginine-rich cathelicidin peptides were purified, including the originally predicted OaBac5 and another OaBac5 variant. As well as this, the C-terminus of the predicted OaBac7.5 and various truncated forms of OaBac11 were purified. Even though these peptides were much smaller than those predicted, they still displayed antimicrobial activity.