Thin-layer chromatography and high-performance liquid chromatography for the assay of fatty acid compositions of individual phospholipids in platelets from non-insulin-dependent diabetes mellitus patients: effect of eicosapentaenoic acid ethyl ester administration

Eight major phospholipids were separated by a TLC method with a one-dimensional developing system without any pretreatment of the plate and the fatty acids incorporated into each phospholipid class were analysed by an improved HPLC method with a simple elution system, which has advantages with respect to resolution and analysis time. The fatty acid compositions of individual phospholipids in platelets were investigated following administration of ethyl cis-5,8,11,14,17-eicosapentaenoate for more than 13 weeks to patients with non-insulin-dependent diabetes mellitus. The cis-5,8,11,14,17-eicosapentaenoic acid compositions of all phospholipid classes were significantly increased with decreasing platelet aggregation rates after the administration. These results suggested that the present method provides the complete separation of individual phospholipids in sufficient amounts to allow fatty acid analysis on the isolated phospholipid moieties.     

Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.     

Structure of the partially unliganded met state of 400 kDa hemoglobin: insights into ligand-induced structural changes of giant hemoglobins

Recent crystallographic studies have revealed the structures of some invertebrate extracellular giant hemoglobins of 3,600 kDa or 400 kDa and their common quaternary structure of dodecameric subassembly composed of four kinds of globin subunits (A1, A2, B1, and B2). These results have provided insight into the mechanisms of their unique functional properties of oxygen binding and sulfide binding. All of these structures were solved with oxygenated or CO-liganded forms at low or moderate resolutions. We have determined the crystal structure of 400 kDa Hb from a polychaete Oligobrachia mashikoi at 1.95 A resolution. The electron densities at higher resolution confirm the existence of an isoform of the B1 subunit because of the inconsistency with the model that was built from the formerly known amino acid sequence. The brownish color of the crystals used in this study and the absorption spectrum from the dissolved crystals strongly indicated that the obtained structure was a ferric met state, whereas complete absence of electron density around the distal heme pockets were observed at the A2, B1, and B2 subunits. We concluded that the obtained structure was in unliganded met forms at three of four globin subunits in the 24mer assembly and in oxygenated forms at the remaining A1 subunits. The partially unliganded structure showed remarkable structural changes at the AB loop regions causing quaternary rearrangements of the EF-dimer structure. In contrast, few changes occurred at the interface regions composed of the E and F helices. These results suggest that the ligand-induced structural changes of Oligobrachia Hb are quite different from those of the well-studied mollusk Hb having the same EF-dimer structure. The structural rearrangements make the dodecameric subassembly form a tighter conformation than those of fully oxygenated or CO-liganded dodecamer structure.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

Ser9 phosphorylation of mitochondrial GSK-3beta is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

The aim of this study was to determine the role of GSK-3beta in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3beta and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 +/- 2.7% to 26.0 +/- 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3beta (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3beta knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3beta (K85R). The level of colocalization of intracellular GSK-3beta with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3beta or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3beta colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3beta knockdown. These results suggest that the suppression of GSK-3beta activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.     

Angiotensin-converting enzyme inhibitors attenuated advanced glycation end products-induced renal tubular hypertrophy via enhancing nitric oxide signaling

Advanced glycation end products (AGE) and angiotensin II were closely correlated with the progression of diabetic nephopathy (DN). Nitric oxide (NO) is a protective mediator of renal tubular hypertrophy in DN. Here, we examined the molecular mechanisms of angiotensin-converting enzyme inhibitor (ACEI) and NO signaling responsible for diminishing AGE-induced renal tubular hypertrophy. In human renal proximal tubular cells, AGE decreased NO production, inducible NOS activity, guanosine 3′,5′-cyclic monophosphate (cGMP) synthesis, and cGMP-dependent protein kinase (PKG) activation. All theses effects of AGE were reversed by treatment with ACEIs (captopril and enalapril), the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the PKG activator 8-para-chlorophenylthio-cGMPs (8-pCPT-cGMPs). In addition, AGE-enhanced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were clearly reduced by captopril, enalapril, SNAP, and 8-pCPT-cGMPs. The abilities of ACEIs and NO/PKG activation to inhibit AGE-induced hypertrophic growth were verified by the observation that captopril, enalapril, SNAP, and 8-pCPT-cGMPs decreased protein levels of fibronectin, p21 ^Waf1/Cip1, and receptor for AGE. The results of the present study suggest that ACEIs significantly reduced AGE-increased ERK/JNK/p38 MAPK activation and renal tubular hypertrophy partly through enhancement of the NO/PKG pathway.     

Triphala,a formulation of traditional Ayurvedic medicine,shows protective effect against X-radiation in HeLa cells

Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations in Ayurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblica officinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigated its activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin, both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygen species (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actions against X-radiation and bleomycin because both agents damage DNA through the generation of ROS. These observations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cells cultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of the molecular basis for the beneficial effects of Triphala.