Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca(2+)] relation yielding Ca(2+)-saturated maximal force (F(max)), myofilament Ca(2+) sensitivity (EC(50)), and cooperativity (Hill coefficient, n(H)) parameters. POLVH was associated with a 35% reduction in F(max) and 36% increase in EC(50). Similarly, MICHF resulted in a 42% reduction in F(max) and a 30% increase in EC(50). Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca(2+) sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC(50) to levels observed in failing myocytes. The F(max) parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca(2+) sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI.     

Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.     

Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.     

Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes

Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.     

Altered alpha1-syntrophin expression in myofibers with Duchenne and Fukuyama muscular dystrophies

Alpha1-syntrophin, a scaffolding adapter and modular protein, is a cytoplasmic component of the dystrophin glycoprotein complex. This study investigated immunohistochemically the expression of alpha1-syntrophin in Duchenne and Fukuyama muscular dystrophies (DMD and FCMD, respectively). Biopsied muscles of five DMD, five FCMD, five normal controls and five disease controls (three myotonic and two facioscapulohumeral dystrophies) were analyzed. Immunoblot analysis showed that anti-alpha1-syntrophin antibody had a decreased reaction in both DMD and FCMD muscle extracts. Biopsied muscle sections and their serial sections were immunostained with rabbit anti-alpha1-syntrophin and rabbit anti-muscle-specific beta-spectrin antibodies, respectively. Immunoreactive patterns of sarcolemma were classified into (i) a continuously positive immunostaining pattern, (ii) a partially positive immunostaining pattern, (iii) a negative immunostaining pattern and (iv) a faint but entire surface positive immunostaining pattern. The group mean percentages of alpha1-syntrophin and beta-spectrin immunonegative myofibers in the DMD group were 39.3% and 10.8%, respectively, while those in the FCMD group were 45.5% and 10.4%, respectively. These values were statistically significant compared with those of disease control and normal control muscles. Thus we found that dystrophin-deficient DMD muscles contained significant numbers of alpha1-syntrophin-positive fibers and significant numbers of alpha1-syntrophin-negative fibers were present in dystrophin-positive muscles of severe muscular dystrophy such as FCMD. Alpha-dystrobrevin immunoreactivity was tested in DMD muscles and appreciable amounts of alpha-dystrobrevin that binds to syntrophin were found in DMD muscle membranes.     

Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1

Members of the HES subfamily of bHLH proteins play crucial roles in neural patterning via repression of neurogenesis. In C. elegans, loss-of-function mutations in ref-1, a distant nematode-specific member of this subfamily, were previously shown to cause ectopic neurogenesis from postembryonic lineages. However, while the vast majority of the nervous system in C. elegans is generated embryonically, the role of REF-1 in regulating these neural lineage decisions is unknown. Here, we show that mutations in ref-1 result in the generation of multiple ectopic neuron types derived from an embryonic neuroblast. In wild-type animals, neurons derived from this sublineage are present in a left/right symmetrical manner. However, in ref-1 mutants, while the ectopically generated neurons exhibit gene expression profiles characteristic of neurons on the left, they are present only on the right side. REF-1 functions in a Notch-independent manner to regulate this ectopic lineage decision. We also demonstrate that loss of REF-1 function results in defective differentiation of an embryonically generated serotonergic neuron type. These results indicate that REF-1 functions in both Notch-dependent and independent pathways to regulate multiple developmental decisions in different neuronal sublineages.     

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Topical application of DEET for schistosomiasis

N, N-diethyl-m-toluamide (also known as DEET) is a broad-spectrum insect repellent that is used extensively against both human and animal pests, worldwide. Recent studies show that topical lipid formulations of DEET, such as LipoDEET, are highly effective in killing schistosome cercariae in the skin. Minimal systemic absorption, low manufacturing cost, and a wide range of activity against insects and schistosomes potentially makes compounds such as LipoDEET an excellent prophylactic agent against human and animal schistosomiasis in endemic areas, especially for travelers, until an effective vaccine is available.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.     

LB3D: a protein three-dimensional substructure search program based on the lower bound of a root mean square deviation value

Searching for protein structure-function relationships using three-dimensional (3D) structural coordinates represents a fundamental approach for determining the function of proteins with unknown functions. Since protein structure databases are rapidly growing in size, the development of a fast search method to find similar protein substructures by comparison of protein 3D structures is essential. In this article, we present a novel protein 3D structure search method to find all substructures with root mean square deviations (RMSDs) to the query structure that are lower than a given threshold value. Our new algorithm runs in O(m + N/m(0.5)) time, after O(N log N) preprocessing, where N is the database size and m is the query length. The new method is 1.8-41.6 times faster than the practically best known O(N) algorithm, according to computational experiments using a huge database (i.e., >20,000,000 C-alpha coordinates).