Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.     

Strain-specific difference in amino acid sequences of trypanosome alternative oxidase

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. The cDNA for TAO has been cloned from Trypanosoma brucei brucei EATRO110 strain and has been used for further characterization. In this study, we found amino acid sequence of the C-terminal part of TAO from the strain that we are using, T. b. brucei TC221, is considerably different from that of the EATRO110 strain.     

Dapagliflozin,a Sodium-Glucose Co-Transporter 2 Inhibitor,Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.     

Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca(2+) influx, membrane pore formation, cell death, and interleukin-1beta release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Antioxidant properties of some different molecular weight chitosans

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6–CT7) with that of low Mr chitosans (CT1–CT5) showed that low Mr chitosans (CT1–CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1–CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the ‘TPAC’ test. These results suggest that low Mr chitosans (CT1–CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.