Identification of genes related to heart failure using global gene expression profiling of human failing myocardium

Although various management methods have been developed for heart failure, it is necessary to investigate the diagnostic or therapeutic targets of heart failure. Accordingly, we have developed different approaches for managing heart failure by using conventional microarray analyses. We analyzed gene expression profiles of myocardial samples from 12 patients with heart failure and constructed datasets of heart failure-associated genes using clinical parameters such as pulmonary artery pressure (PAP) and ejection fraction (EF). From these 12 genes, we selected four genes with high expression levels in the heart, and examined their novelty by performing a literature-based search. In addition, we included four G-protein-coupled receptor (GPCR)-encoding genes, three enzyme-encoding genes, and one ion-channel protein-encoding gene to identify a drug target for heart failure using in silico microarray database. After the in vitro functional screening using adenovirus transfections of 12 genes into rat cardiomyocytes, we generated gene-targeting mice of five candidate genes, namely, MYLK3, GPR37L1, GPR35, MMP23, and NBC1. The results revealed that systolic blood pressure differed significantly between GPR35-KO and GPR35-WT mice as well as between GPR37L1-Tg and GPR37L1-KO mice. Further, the heart weight/body weight ratio between MYLK3-Tg and MYLK3-WT mice and between GPR37L1-Tg and GPR37L1-KO mice differed significantly. Hence, microarray analysis combined with clinical parameters can be an effective method to identify novel therapeutic targets for the prevention or management of heart failure.     

Comparing Symbiotic Efficiency between Swollen versus Nonswollen Rhizobial Bacteroids

Symbiotic rhizobia differentiate physiologically and morphologically into nitrogen-fixing bacteroids inside legume host nodules. The differentiation is apparently terminal in some legume species, such as peas (Pisum sativum) and peanuts (Arachis hypogaea), likely due to extreme cell swelling induced by the host. In other legume species, such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata), differentiation into bacteroids, which are similar in size and shape to free-living rhizobia, is reversible. Bacteroid modification by plants may affect the effectiveness of the symbiosis. Here, we compare symbiotic efficiency of rhizobia in two different hosts where the rhizobia differentiate into swollen nonreproductive bacteroids in one host and remain nonswollen and reproductive in the other. Two such dual-host strains were tested: Rhizobium leguminosarum A34 in peas and beans and Bradyrhizobium sp. 32H1 in peanuts and cowpeas. In both comparisons, swollen bacteroids conferred more net host benefit by two measures: return on nodule construction cost (plant growth per gram nodule growth) and nitrogen fixation efficiency (H₂ production by nitrogenase per CO₂ respired). Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.Legume-rhizobia interactions vary widely across a diverse paraphyletic group of soil bacteria known for symbiotic nitrogen fixation inside root nodules of over 18,000 species of legumes throughout the world (Lewis et al., 2005). In several legume species, rhizobial cells are induced to swell during their differentiation into nitrogen-fixing bacteroids (Oono et al., 2010). These legume species belong to five different major papilionoid clades (inverted repeat-lacking clade, genistoids, dalbergioids, mirbelioids, and millettioids), a pattern suggestive of convergent evolution. Swelling apparently leads to terminal differentiation; swollen bacteroids no longer divide normally (Zhou et al., 1985). In other legume host species, bacteroid differentiation is less extreme, leading to nonswollen bacteroids. Nonswollen bacteroids are similar in shape and size to free-living rhizobia and divide normally once outside of their nodules. The proximate mechanisms for host-imposed bacteroid swelling have been investigated (Van de Velde et al., 2010), but what drove the repeated evolution of this trait? The multiple independent origins of host traits causing bacteroids to swell suggest that swollen bacteroids may provide more net benefit to legumes. Could the swelling of bacteroids improve nitrogen fixation efficiency (e.g. nitrogen fixed relative to carbon cost)? In this study, we compare symbiotic efficiencies of rhizobia in legume hosts that are evolutionarily diverged but share a common effective rhizobial strain, whose bacteroids are swollen in one host and nonswollen in the other.Variations among host species in benefits and costs of symbiosis with rhizobia are not commonly explored (Thrall et al., 2000) because legume species typically nodulate with only one group of rhizobia (e.g. Sinorhizobium sp. in Medicago), although some legumes and some rhizobia are more promiscuous. Rhizobium sp. NGR234 has the largest known host range but does not fix nitrogen effectively with any legume species currently recognized to induce swelling of rhizobial bacteroids (Pueppke and Broughton, 1999). Some Sinorhizobium fredii strains apparently fix nitrogen in certain cultivars of soybean (Glycine max; hosting nonswollen bacteroids) and alfalfa (Medicago sativa; hosting swollen bacteroids; Hashem et al., 1997), but our efforts to replicate these results did not lead to successful nodulation. Therefore, we studied two strains, a transgenic strain that nodulates beans (Phaseolus vulgaris) and peas (Pisum sativum) and a second wild strain harvested from cowpeas (Vigna unguiculata) that also nodulates peanuts (Arachis hypogaea). Beans and cowpeas are both within the Phaseolid group and do not induce terminal differentiation of rhizobial bacteroids. Peas and peanuts both host terminally differentiated bacteroids but are in distant clades and likely have different genetic origins for traits that induce terminal differentiation (Oono et al., 2010). Also, the swollen bacteroids in peas are branched while those in peanuts are spherical.Differences in symbiotic qualities between swollen and nonswollen bacteroids have been previously explored in peanuts and cowpeas by Sen and Weaver (1980, 1981, 1984), who also hypothesized that swollen bacteroids are more beneficial to the host plant than nonswollen ones. They found 1.5 to 3 times greater acetylene reduction by nitrogenase (as well as plant nitrogen) per nodule mass in peanuts than in cowpeas at multiple nodule ages (Sen and Weaver, 1980). Acetylene reduction per bacteroid was also greater in peanuts than in cowpeas when measuring whole nodules, but this difference disappeared when isolated bacteroids were assayed (Sen and Weaver, 1984). They concluded that swelling of peanut bacteroids per se was not responsible for the higher rate of nitrogen fixation per bacteroid. They suggested that in cowpea nodules, with greater numbers of smaller bacteroids per nodule volume, availability of oxygen to each bacteroid might be restricted such that the rate of oxidative phosphorylation, necessary for nitrogen fixation, is reduced. Fixation rates per bacteroid may be different between hosts due to nodule gas permeability or bacteroid crowding within nodules. However, fixation efficiency (nitrogen fixed per carbon respired) would not necessarily be affected by these and may be more important for the host than the rate of fixation.Rhizobial performances are often compared by measuring the symbiotic benefits, e.g. rates of acetylene reduction or plant growth (Sen and Weaver, 1984; Hashem et al., 1997; Lodwig et al., 2005), but rarely by measuring the symbiotic costs, e.g. carbon consumed or respired. Up to 25% of a legume’s net photosynthate may be required for nitrogen fixation by rhizobia (Minchin et al., 1981). Faster fixation rates (mol nitrogen per s) can be beneficial for hosts, but carbon costs can also be important. Rhizobia that fix more nitrogen per carbon respired could free more carbon for other functions, including the option of supporting more nodules with the same amount of photosynthate. If legumes are sometimes carbon limited, then improved carbon-use efficiency could enhance plant fitness. Measuring both benefits and costs is therefore key to an accurate understanding of the symbiotic performance of a rhizobial strain.While we recognize the many physiological differences between peas and beans or peanuts and cowpeas, the fact that terminal differentiation induced by host legumes evolved multiple times independently (Oono et al., 2010) suggests there may be some consistent host symbiotic benefit, such as improved fixation efficiency. Here, we measured the efficiency of each of two strains as swollen bacteroids in one host and nonswollen bacteroids in another. We measured nitrogenase activity as hydrogen (H₂) production in an N₂-free atmosphere (Layzell et al., 1984; Witty and Minchin, 1998), and compared it to carbon dioxide (CO₂) respiration to estimate return on nodule operation cost. We also compared host biomass growth per total nodule mass growth to estimate return on nodule construction cost. To further assess carbon allocation to the different types of bacteroids, we also measured the average amounts per bacteroid of polyhydroxybutyrate (PHB), an energy storage compound that can comprise up to 50% of bacteroid dry weight (Trainer and Charles, 2006). A greater PHB accumulation per bacteroid may require a decreased allocation of carbon for nitrogenase activity within the bacteroids, and hence, less plant growth per carbon invested in bacteroids. We demonstrate that peas and peanuts that host swollen bacteroids have higher fixation efficiency as well as greater plant return on nodule construction than beans and cowpeas, respectively, nodulated with the same rhizobial strains. PHB was not consistently correlated with plant:nodule growth efficiency with the tested strains. These findings show that swollen bacteroids can indeed provide greater benefits to their legume hosts.     

Genetic diversity of gamma-hexachlorocyclohexane-degrading sphingomonads isolated from a single experimental field

Aim:  To determine the effect of the surface roughness of denture acrylic on the attachment of Streptococcus oralis .
Methods and Results:  Roughened denture acrylic samples were assessed for bacterial attachment, over time, using microscopy. The area of the image covered by bacteria was calculated and converted into a percentage of the total area sampled. The results showed an increasing bacterial coverage with time of incubation and increasing roughness. Differences were seen between heat cured acrylic and cold cured acrylic.
Conclusion:  This study successfully demonstrated a system for the assessment of the amount of attached bacteria on denture acrylic varying roughness. The system was able to discern the difference in surface area coverage by attached bacteria over a roughness range relevant to brushing dentures with dentifrices.
Significance and Impact of Study:  This study provides strong support for the scratches caused by brushing dentures with dentifrice encouraging bacterial attachment. This is likely to have a significant effect on efficacy of denture cleaning, general hygiene and biofilm re-formation between cleaning regimens and may indicate that alternative low abrasive cleaners, such as antimicrobial denture-cleaning tablets, offer a more appropriate regimen.     

DEVELOPMENTAL STUDY OF BRANCHED RHIZOPHORES IN THREE SELAGINELLA SPECIES

A morphogenetic investigation was made of the rhizophore of three large-sized tropical Selaginella species. The rhizophores of Selaginella delicatula, S. caudata, and S. plana arise exogenously at the points of branching of the main stems. In S. delicatula they are initiated at the junction of the second youngest branching. The rhizophore apical meristem has a tetrahedral apical cell and is capless. The rhizophores are usually three or four times dichotomously branched in S. delicatula and S. plana and four or five times in S. caudata. In S. delicatula, dichotomous branching of the rhizophore involves formation of two new apical cells subsequent to loss of an original apical cell. A pair of roots is formed endogenously from inner cells below the dermal layer at the apex of ultimate rhizophore branches. The finding that the rhizophore is an autonomously branched, leafless, and capless axis leads us to argue that Selaginella rhizophores, like lepidodendrid rhizomorphs, are fundamental axial organs that coordinate with the stem and root.     

Effects of compressive force on the differentiation of pluripotent mesenchymal cells

The purpose of this study was to determine the effect of mechanical stress on the differentiation of the pluripotent mesenchymal cell line C2C12. C2C12 cells were cultured continuously under compressive force (0.25-2.0 g/cm(2)). After mechanical stress loading, the levels of expression of mRNAs and proteins for phenotype-specific markers of osteoblasts (Runx2, Msx2, Dlx5, Osterix, AJ18), chondroblasts (Sox5, Sox9), myoblasts (MyoD), and adipocytes (PPAR gamma) were measured by real-time polymerase chain reaction analysis and Western blot analysis, respectively. The expression of activated p38 mitogen-activated protein kinase (p38 MAPK) was measured by Western blotting and/or ELISA. Loading 0.5 g/cm(2) of compressive force significantly increased the expression levels of Runx2, Msx2, Dlx5, Osterix, Sox5, and Sox9. In contrast, the expression levels of AJ18, MyoD, and PPAR gamma were decreased by exposure to 0.5 g/cm(2) of compressive force. Loading 0.5 g/cm(2) of compressive force also induced the phosphorylation of p38 MAPK. SB203580, which is a specific inhibitor of p38 MAPK, inhibited the compressive force-induced phosphorylation of p38 MAPK and partially blocked compressive force-induced Runx2 mRNA expression. These results demonstrate that compressive force stimulation directs the differentiation pathway of C2C12 cells into the osteoblast and chondroblast lineage via activated phosphorylation of p38 MAPK.     

Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.     

Evolution of shoot apical meristem structures in vascular plants with respect to plasmodesmatal network

Vascular plants have evolved shoot apical meristems (SAMs), whose structures differ among plant groups. To clarify the evolutionary course of the different structural types of SAMs, we compared plasmodesmatal networks in the SAMs for 17 families and 24 species of angiosperms, gymnosperms, and pteridophytes, using transmission electron microscopy (TEM). The plasmodesmata (PD) in almost all cell walls in median longitudinal sections of SAMs were counted, and the PD density per unit area was calculated for each cell wall. Angiosperm and gymnosperm SAMs have low densities, with no difference between stratified (tunica-corpus) and unstratified structures. SAMs of ferns, including Psilotum and Equisetum, have average densities that are more than three times higher than those of seed plants. Interestingly, microphyllous lycopods have both the fern and seed-plant types of PD networks; Selaginellaceae SAMs with single apical cells have high PD densities, while SAMs of Lycopodiaceae and Isoetaceae with plural initial cells have low PD densities, equivalent to those of seed plants. In summary, PD networks are strongly correlated to SAM organizations-SAMs with single and plural initial cells have the fern and seed-plant types of PD, respectively. The two SAM organizations may have evolved separately in lycophytes and euphyllophytes and may be associated with gain or loss of the ability to form secondary PD.     

Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2

Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.