ANG II enhances contractile responses via PI3-kinase p110 delta pathway in aortas from diabetic rats with systemic hyperinsulinemia

We investigated the involvement of ANG II and phosphatidylinositol 3-kinase (PI3-K) in the enhanced aortic contractile responses induced by hyperinsulinemia in chronic insulin-treated Type 1 diabetic rats. Plasma ANG II levels were elevated in untreated compared with control diabetic rats and further increased in insulin-treated diabetic rats. Aortic contractile responses and systolic blood pressure were significantly enhanced in chronic insulin-treated diabetic rats compared with the other groups. These insulin-induced increases were largely prevented by cotreatment with losartan (an ANG II type 1 receptor antagonist) or enalapril (an angiotensin-converting enzyme inhibitor). LY-294002 (a PI3-K inhibitor) diminished the increases in contractile responses in ANG II-incubated aortas and aortas from chronic insulin-treated diabetic rats. The norepinephrine (NE)-stimulated levels of p110 delta-associated PI3-K activity and p110 delta protein expression were increased in aortas from insulin-treated diabetic compared with control and untreated diabetic rats, and chronic administration of losartan blunted these increases. Contractions were significantly larger in aortas from diabetic rats incubated with a low concentration (inducing approximately 10% of the maximum contraction) of ANG II or with NE or isotonic K+ than in aortas from nonincubated diabetic rats. NE-stimulated p110 PI3-K activity was elevated in aortas from diabetic rats coincubated with a noncontractile dose of ANG II. These results suggest that, in insulin-treated Type 1 diabetic rats with hyperinsulinemia, chronic ANG II type 1 receptor blockade blunts the increases in vascular contractility and blood pressure via a decrease in p110 delta-associated PI3-K activity.     

Structural requirements for activation in alphaIIb beta3 integrin

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.     

Genome Sequence of Kitasatospora setae NBRC 14216T: An Evolutionary Snapshot of the Family Streptomycetaceae

Kitasatospora setae NBRC 14216^T (=KM-6054^T) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC 14216^T as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8 783 278 bp with terminal inverted repeats of 127 148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.     

CX3CL1 expression induced by Candida albicans in oral fibroblasts

Oral fibroblasts as well as keratinocytes are thought to influence host inflammatory responses against Candida albicans. However, little is known about chemokine expressions in oral fibroblasts against C. albicans infection. We therefore examined whether C. albicans induced several chemokines including fractalkine/CX3CL1 (CX3CL1), a unique chemokine that has properties of both chemoattractants and adhesion molecules, in fibroblasts and keratinocytes. The addition of C. albicans live cells to human immortalized oral keratinocytes (RT7) resulted in increases in the mRNA levels of multiple chemokines, but not of CX3CL1. In contrast, live and heat-killed C. albicans caused an increase in CX3CL1 mRNA and protein expression in human immortalized oral fibroblasts (GT1). CX3CL1 mRNA expression in GT1 cells was also enhanced by stimulation with a nonalbicans species of Candida. Further, the CX3CL1 chemokine domain showed antifungal activity against C. albicans. CX3CL1 secreted by oral fibroblasts appears to play an important role in the oral immune response to C. albicans infection.     

Expansion of the trans-Golgi network following activated collagen secretion is supported by a coiled-coil microtubule-bundling protein, p180, on the ER

A coiled-coil endoplasmic reticulum (ER) protein, p180, was originally reported as a ribosome-binding receptor on the rough ER and is highly expressed in secretory tissues. Recently, we reported new functions of p180 as a microtubule-bundling protein on the ER. Here, we investigated the specific roles of p180 in the Golgi complex organization following stimulated collagen secretion. Targeted depletion of p180 by siRNA transfection caused marked reduction of TGN, while other marker levels for the cis or medial Golgi were not markedly changed. Ascorbate stimulation resulted in trans-Golgi network (TGN) expansion to the periphery in control cells that is characterized by both increased membrane amounts and extended shape. In contrast, loss of p180 resulted in retraction of the TGN regardless of ascorbate stimulation. The TGN developed to the periphery along stabilized microtubule bundles, and overexpression of MTB-1 fragment caused dominant-negative phenotypes. Once disorganized, the retracted TGN did not recover in the absence of p180 despite elevated acetylated tubulin levels. TGN46 and p180 were co-distributed in epithelial basal layer cells of human mucosal and gastrointestinal tissues. Taken together, we propose a novel function of p180-abundant ER on the TGN expansion, both of which are highly developed in various professional secretory cells.     

Metformin normalizes endothelial function by suppressing vasoconstrictor prostanoids in mesenteric arteries from OLETF rats, a model of type 2 diabetes

We previously reported that in mesenteric arteries from aged Otsuka Long-Evans Tokushima fatty (OLETF) rats (a type 2 diabetes model) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired while endothelium-derived contracting factor (EDCF)-mediated contraction is enhanced (Matsumoto T, Kakami M, Noguchi E, Kobayashi T, Kamata K. Am J Physiol Heart Circ Physiol 293: H1480-H1490, 2007). Here we investigated whether acute and/or chronic treatment with metformin might improve this imbalance between the effects of the above endothelium-derived factors in mesenteric arteries isolated from OLETF rats. In acute studies on OLETF mesenteric arteries, ACh-induced relaxation was impaired and the relaxation became weaker at high ACh concentrations. Both metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, an AMP-activated protein kinase (AMPK) activator that is also activated by metformin] 1) diminished the tendency for the relaxation to reverse at high ACh concentrations and 2) suppressed both ACh-induced EDCF-mediated contraction and ACh-stimulated production of prostanoids (thromboxane A2 and PGE2). In studies on OLETF arteries from chronically treated animals, metformin treatment (300 mg.kg(-1).day(-1) for 4 wk) 1) improved ACh-induced nitric oxide- or EDHF-mediated relaxation and cyclooxygenase (COX)-mediated contraction, 2) reduced EDCF-mediated contraction, 3) suppressed production of prostanoids, and 4) reduced superoxide generation. Metformin did not alter the protein expressions of endothelial nitric oxide synthase (eNOS), phospho-eNOS (Ser1177), or COX-1, but it increased COX-2 protein. These results suggest that metformin improves endothelial functions in OLETF mesenteric arteries by suppressing vasoconstrictor prostanoids and by reducing oxidative stress. Our data suggest that within the timescale studied here, metformin improves endothelial function through this direct mechanism, rather than by improving metabolic abnormalities.     

Effects of Ca2+ on flavin-linked complex enzymes in mitochondria isolated from eggs and embryos of sea urchin

Mitochondria isolated from sea urchin embryos in early development show almost the same activities of cytochrome c oxidase and flavin-linked complex enzymes, which are estimated by cytochrome c reductases as in those isolated from unfertilized eggs. The activities of these cytochrome c reductases are inhibited by Ca2+ at above 10-5 M more strongly than cytochrome c oxidase. To investigate the changes in intramitochondrial Ca2+ concentration at fertilization, the activity of pyruvate dehydrogenase, another mitochondrial enzyme, was measured. The activity of this enzyme was controlled by phosphorylation and Ca2+-dependent dephosphorylation of the catalytic unit. The enzyme activity increased for 30 min after fertilization, decreased and became close to zero within ~60 min. Then, the activity appreciably increased again after hatching. This seems to reflect changes in the intramitochondrial Ca2+ concentration. The enzyme activity was enhanced by pre-incubation with Ca2+ at concentrations up to 10-5 M but was made quite low at above 10-4 M Ca2+ and 10-3 M adenosine triphosphate. Although the changes in pyruvate dehydrogenase activity observed at fertilization will reflect the changes in the intramitochondrial calcium concentration, the intramitochondrial Ca2+ concentration of unfertilized eggs cannot be estimated from these results because high (> 10-4 M) or low (10-6 M) Ca2+ can inhibit the enzyme. Measurement of respiration of a single egg showed that injection of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid released the mitochondrial electron transport in the unfertilized egg. The possibility that changes in intramitochondrial calcium concentration occur at fertilization is discussed in relation to activation of both mitochondrial respiration and pyruvate dehydrogenase.     

A carboxy-terminally truncated form of the human immunodeficiency virus type 1 Vpr protein induces apoptosis via G(1) cell cycle arrest

Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G(2) phase and inducing to apoptosis after G(2) arrest. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G(2) arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce apoptosis via G(1) arrest of the cell cycle. Immunostaining for various markers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C81 expression vector were arrested at the G(1) phase and not at the G(2) or S phase of the cell cycle. Staining for annexin V, which binds phosphatidylserine on the plasma membrane, as an early indicator of apoptosis and measurement of the activity of caspase-3, a signaling molecule in apoptotic pathways, indicated that C81 is a strong inducer of apoptosis. Expression of C81 induced the condensation, fragmentation, and clumping of chromatin that are typical of apoptosis. Furthermore, the kinetics of the C81-induced G(1) arrest were closely correlated with changes in the number of annexin V-positive cells and the activity of caspase-3. Replacement of Ile or Leu residues by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G(1) arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized for G(1) arrest of the cell cycle. It has been reported that Ile60, Leu67, and Ile74 also play an important role in the C81-induced suppression of growth. These results suggest that the suppression of growth induced by C81 result in apoptosis that is independent of G(2) arrest of the cell cycle.     

Mild irritant prevents ethanol-induced gastric mucosal microcirculatory disturbances through actions of calcitonin gene-related peptide and PGI2 in rats

Pretreatment with a mild irritant such as 1 M NaCl prevented ethanol-induced mucosal injury, which was abolished by indomethacin, suggesting involvement of endogenous PGs. With the use of intravital microscopy, we investigated the mechanism in microcirculation whereby a mild irritant prevents ethanol-induced mucosal injury. Microcirculation of the basal part of gastric mucosa in anesthetized rats was observed through a window with transillumination. Diameters of arterioles, collecting venules, and venules were measured with an electric microscaler. One molar NaCl alone caused dilation of arterioles and constrictions of collecting venules and venules, which were inhibited by indomethacin. Ethanol (50%) applied to mucosa constricted collecting venules and venules but dilated arterioles. Constriction of collecting venules resulted in mucosal congestion. Pretreatment with 1 M NaCl inhibited ethanol-induced constrictions of collecting venules and venules, and administration of indomethacin or a calcitonin gene-related peptide (CGRP) antagonist, CGRP-(8-37), abolished elimination of constrictions. Topical application (1 nM-10 microM) of PGE2 or beraprost sodium (a PGI2 analog) to microvasculature markedly and dose-dependently dilated arterioles, whereas that of PGE2, but not beraprost, slightly constricted collecting venules. Pretreatment of microvasculature with a nonvasoactive concentration of PGE2 (100 nM) or beraprost (1 nM) completely inhibited ethanol-induced constriction of collecting venules. The inhibitory effect of beraprost but not of PGE2 was abolished by CGRP-(8-37). Present results suggest that the mechanism whereby 1 M NaCl prevents ethanol-induced injury is elimination of constrictions of collecting venules and venules by CGRP whose release may be enhanced by PGI2 but not by PGE2.     

Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen

We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.