p53-induced adipose tissue inflammation is critically involved in the development of insulin resistance in heart failure

Several clinical studies have shown that insulin resistance is prevalent among patients with heart failure, but the underlying mechanisms have not been fully elucidated. Here, we report a mechanism of insulin resistance associated with heart failure that involves upregulation of p53 in adipose tissue. We found that pressure overload markedly upregulated p53 expression in adipose tissue along with an increase of adipose tissue inflammation. Chronic pressure overload accelerated lipolysis in adipose tissue. In the presence of pressure overload, inhibition of lipolysis by sympathetic denervation significantly downregulated adipose p53 expression and inflammation, thereby improving insulin resistance. Likewise, disruption of p53 activation in adipose tissue attenuated inflammation and improved insulin resistance but also ameliorated cardiac dysfunction induced by chronic pressure overload. These results indicate that chronic pressure overload upregulates adipose tissue p53 by promoting lipolysis via the sympathetic nervous system, leading to an inflammatory response of adipose tissue and insulin resistance.     

Phytochemical and biological investigation of Hymenocallis littoralis SALISB

A phytochemical investigation of the bulbs and flowers of Hymenocallis littoralis SALISB., cultivated in Egypt, was carried out, which resulted in the isolation of four alkaloids, lycorine (1), hippeastrine (2), 11-hydroxyvittatine (3), and (+)-8-O-demethylmaritidine (4), and of two flavonoids, quercetin 3'-O-glucoside (5), and rutin (6). The volatile constituents of the plant flowers were analyzed for the first time by GC/MS, which led to the identification of 26 known compounds (Table 1). Finally, the antimicrobial activity of the petroleum ether extract of the flowers of H. littoralis was investigated.     

High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81

The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8+/-1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins.     

Identification of a novel alpha(1-->6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant

Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C(16)/C(18:1)-(alpha-D-glucopyranosyluronic acid)-(1-->3)-glycerol (GlcAGroAc(2)) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) and Man(1)GlcAGroAc(2) were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[(14)C]Man established that C. glutamicum synthesized a novel alpha(1-->6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac(1)PIM(2) and Man(1)GlcAGroAc(2) respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core alpha(1-->6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac(1)PI[(14)C]M(2) and [(14)C]Man(1)GlcAGroAc(2) primers respectively. Use of the acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C(8) in an in vitro cell-free assay confirmed NCgl1505 as an alpha(1-->6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroalpha(1-->6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.     

Possible Association of GroES and Antigen 85 Proteins with Heat Resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D_65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D_65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major.

We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.     

Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.     

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.