Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits

Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the selectivity of the actions of CaMKII among Ca2+ channel beta subunit isoforms. CaMKII phosphorylates the beta1b, beta2a, beta3, and beta4 isoforms with similar initial rates and final stoichiometries of 6-12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to beta1b and beta2a with a similar apparent affinity but does not bind to beta3 or beta4. Prephosphorylation of beta1b and beta2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in beta2a are highly conserved in beta1b but are different in beta3 and beta4. Site-directed mutagenesis of this domain in beta2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-beta2a complexes in vitro and reduces interactions of CaMKII with beta2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with beta2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca2+ channel beta subunits with CaMKII.     

Simultaneous phytoremediation of tannery effluent and production of fatty acids rich biomass by Chlorella sorokiniana

Journal of Applied Phycology - Tanning is a technique of transforming hides to skins and the production of leather products involves a wide variety of processes that produce substantial amounts of...     

Aerobic mineralization of 4,4′-dichlorobiphenyl and 4-chlorobenzoic acid by a novel natural bacterial strain that grows poorly on benzoate and biphenyl

Achromobacter xylosoxidans strain IR08 was isolated from soil contaminated with electrical transformer fluid by enrichment culture containing Aroclor 1221 as the sole carbon source. This strain was found to grow on all monochlorobiphenyls, 4,4′-dichlorobiphenyl (4,4′-diCB) and a wide range of other xenobiotic compounds. During growth on 4,4′-diCB, a near-stoichiometric amount of chloride was excreted into the culture fluid in less than 5 days and growth yield was more than twice that achieved on biphenyl. The production of 4-CBA or chlorocatechol as a metabolite was not observed. Quite unusually, coincubation of strain IR08 with 4,4′-diCB and biphenyl at relatively equal concentrations showed preferential utilization of the chlorobiphenyl: 4,4′-diCB was mineralized in less than 5 days concomitant with stoichiometric release of chloride, while biphenyl was poorly degraded. Growth on 2.5 mM CBA also resulted in complete disappearance of the substrate, however, inorganic chloride recovered from the culture broth was less than 65%. The isolation of a dichlorobiphenyl-mineralizing rather than transformation strain such as IR08 is an important advance in an effort to develop effective bioremediation strategy for polychlorinated biphenyl-contaminated soil.     

Importance of timing of application of the entomopathogenic fungus,metarhizium anisopliae for the control of legume flower thrips,megalurothrips sjostedti and its persistence on cowpea

Field experiments were conducted for two seasons to evaluate the timing of application of the entomopathogenic fungus, Metarhizium anisopliae for the control of legume flower thrips, Megalurothrips sjostedti on cowpea. One application of M. unisopliae timed at flower bud stage and another at flowering stage did not protect cowpea yield against M. sjostedti as does chemical insecticide, Karate (Lambda‐cyhalothrin). Instead, one application of the fungus given at flower bud stage and two applications given at flowering were required to keep M. sjostedti population in check through these stages, which are very sensitive to thrips damage with a concomitant increase in cowpea yield which was significantly higher than the Karate treatment. Studies of persistence showed that M. anisopliae remained active in the field for 3–4 days.     

Potent peptide inhibitors of smooth muscle myosin light chain kinase: mapping of the pseudosubstrate and calmodulin binding domains

Smooth muscle myosin light chain kinase (MLCK) is activated by calcium-calmodulin and, in turn, phosphorylates and activates the smooth muscle actomyosin ATPase, resulting in muscle contraction. The amino acid sequence of the regulatory domain of MLCK is known, and it contains a region that binds calmodulin and also bears a strong homology to the phosphorylation site in the substrate. Thus, it has been called the "pseudosubstrate". It has been proposed that calmodulin activates MLCK by binding to and reversing the autoinhibitory function of the pseudosubstrate. Synthetic peptides based on this sequence inhibit MLCK both by binding to calmodulin and by competing with the substrate at the active site. In the work reported here, we have synthesized a large number of peptides from the regulatory region of MLCK (MLCK 480-516). The region was systematically analyzed by dividing it into fragments of two to six amino acids, each containing one or more basic residues, in order to map in detail the calmodulin binding site and the autoinhibitory region. It was observed that both calmodulin binding and autoinhibition are mediated by several different fragments of the regulatory sequence. Two nonoverlapping peptides, MLCK 480-493 and MLCK 494-504, are similar in potency in inhibiting the enzyme (IC50's of 2 and 6 microM, respectively). Larger fragments, combining multiple inhibitory regions, are more potent inhibitors. For example, MLCK 480-504 is extremely potent, with an IC50 of 13 nM. The calmodulin binding site and active site directed inhibitory regions overlap, but are not identical. Residues 505-512 are important only for calmodulin binding.     

Myocardial ischaemia inhibits mitochondrial metabolism of 4-hydroxy-trans-2-nonenal

Myocardial ischaemia is associated with the generation of lipid peroxidation products such as HNE (4-hydroxy-trans-2-nonenal); however, the processes that predispose the ischaemic heart to toxicity by HNE and related species are not well understood. In the present study, we examined HNE metabolism in isolated aerobic and ischaemic rat hearts. In aerobic hearts, the reagent [(3)H]HNE was glutathiolated, oxidized to [(3)H]4-hydroxynonenoic acid, and reduced to [(3)H]1,4-dihydroxynonene. In ischaemic hearts, [(3)H]4-hydroxynonenoic acid formation was inhibited and higher levels of [(3)H]1,4-dihydroxynonene and [(3)H]GS-HNE (glutathione conjugate of HNE) were generated. Metabolism of [(3)H]HNE to [(3)H]4-hydroxynonenoic acid was restored upon reperfusion. Reperfused hearts were more efficient at metabolizing HNE than non-ischaemic hearts. Ischaemia increased the myocardial levels of endogenous HNE and 1,4-dihydroxynonene, but not 4-hydroxynonenoic acid. Isolated cardiac mitochondria metabolized [(3)H]HNE primarily to [(3)H]4-hydroxynonenoic acid and minimally to [(3)H]1,4-dihydroxynonene and [(3)H]GS-HNE. Moreover, [(3)H]4-hydroxynonenoic acid was extruded from mitochondria, whereas other [(3)H]HNE metabolites were retained in the matrix. Mitochondria isolated from ischaemic hearts were found to contain 2-fold higher levels of protein-bound HNE than the cytosol, as well as increased [(3)H]GS-HNE and [(3)H]1,4-dihydroxynonene, but not [(3)H]4-hydroxynonenoic acid. Mitochondrial HNE oxidation was inhibited at an NAD(+)/NADH ratio of 0.4 (equivalent to the ischaemic heart) and restored at an NAD(+)/NADH ratio of 8.6 (equivalent to the reperfused heart). These results suggest that HNE metabolism is inhibited during myocardial ischaemia owing to NAD(+) depletion. This decrease in mitochondrial metabolism of lipid peroxidation products and the inability of the mitochondria to extrude HNE metabolites could contribute to myocardial ischaemia/reperfusion injury.     

Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury

Capillary leakage and alveolar edema are hallmarks of acute lung injury (ALI). Neutrophils and serum macromolecules enter alveoli, promoting inflammation. Vascular endothelial growth factor (VEGF) causes plasma leakage in extrapulmonary vessels. Angiopoietin (Ang)-1 and -4 stabilize vessels, attenuating capillary leakage. We hypothesized that VEGF and Ang-1 and -4 modulate vessel leakage in the lung, contributing to the pathogenesis of ALI. We examined a murine model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 and 129/J mice were studied at baseline and 24, 48, and 96 h after single or multiple doses of aerosolized LPS. Both strains exhibited time- and dose-dependent increases in inflammation and a deterioration of lung mechanics. Bronchoalveolar lavage (BAL) protein levels increased significantly, suggesting capillary leakage. Increased BAL neutrophil and total protein content correlated with time-dependent increased tissue VEGF and decreased Ang-1 and -4 levels, with peak VEGF and minimum Ang-1 and -4 expression after 96 h of LPS challenge. These data suggest that changes in the balance between VEGF and Ang-1 and -4 after LPS exposure may modulate neutrophil influx, protein leakage, and alveolar flooding during early ALI.     

Reconstructing the history of maize streak virus strain a dispersal to reveal diversification hot spots and its origin in southern Africa

Maize streak virus strain A (MSV-A), the causal agent of maize streak disease, is today one of the most serious biotic threats to African food security. Determining where MSV-A originated and how it spread transcontinentally could yield valuable insights into its historical emergence as a crop pathogen. Similarly, determining where the major extant MSV-A lineages arose could identify geographical hot spots of MSV evolution. Here, we use model-based phylogeographic analyses of 353 fully sequenced MSV-A isolates to reconstruct a plausible history of MSV-A movements over the past 150 years. We show that since the probable emergence of MSV-A in southern Africa around 1863, the virus spread transcontinentally at an average rate of 32.5 km/year (95% highest probability density interval, 15.6 to 51.6 km/year). Using distinctive patterns of nucleotide variation caused by 20 unique intra-MSV-A recombination events, we tentatively classified the MSV-A isolates into 24 easily discernible lineages. Despite many of these lineages displaying distinct geographical distributions, it is apparent that almost all have emerged within the past 4 decades from either southern or east-central Africa. Collectively, our results suggest that regular analysis of MSV-A genomes within these diversification hot spots could be used to monitor the emergence of future MSV-A lineages that could affect maize cultivation in Africa.