Hybrid respiration in the denitrifying mitochondria of Fusarium oxysporum

Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)). Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors. The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase. We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria.     

Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants

Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.     

Determination of free N-acetylneuraminic acid in urine by high-performance liquid chromatography using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent

A highly sensitive high-performance liquid chromatography (HPLC) method for the determination of urinary N-acetylneuraminic acid (NeuAc) using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent was developed. The labeling reaction was carried out at 30 degrees C for 30 min in the presence of pyridine. The derivative was monitored at Ex 314 nm and Em 388 nm. The detection limit of NeuAc was about 48 fmol per injection. The relative standard deviations of within-day and between-day precisions were 2.6-3.3 and 1.7-3.3%, respectively. Urine diluted 10 times with distilled water was analyzed by employing the standard-addition method. The concentrations were 8-89 nmol/mg creatinine (30+/-28 nmol/mg creatinine, n=9).     

Oligosaccharides as receptors for JC virus

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including alpha1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on alpha2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal alpha2-3- or alpha2-6-linked sialic acid or the branched alpha2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal alpha2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal alpha2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.     

Characterization of radioiodinated (-)-ortho-iodovesamicol binding in rat brain preparations

We investigated the binding characteristics of optical isomers of three iodovesamicol analogs to vesicular acetylcholine transporters (VAChT) and to sigma receptors (sigma-1, sigma-2) in rat brains. In competitive inhibition studies, (-)-enantiomers [(-)-ortho-iodovesamicol ((-)-oIV), (-)-meta-iodovesamicol ((-)-mIV), (-)-vesamicol] displayed a higher affinity for VAChT than (+)-enantiomer [(+)-oIV, (+)-mIV, (+)-vesamicol]. (-)-oIV and (-)-mIV showed the same high affinity for VAChT as (-)-vesamicol. For sigma receptors(sigma-1, sigma-2), (-)-oIV (Ki = 62.2 nM (to sigma-1) and 554 nM(to sigma-2)) showed a lower affinity than (-)-mIV (Ki = 4.5 nM (to sigma-1) and 42.9 nM (to sigma-2)). Furthermore, in a saturation binding study, (-)-[125I]-oIV exhibited a Kd of 17.4 +/- 5.1 nM with a maximum number of binding sites, Bmax, of 559 +/- 51 fmol/ mg of protein. These results showed that (-)-oIV binds to vesicular acetylcholine transporters (VAChT) more selectively than (-)-mIV, previously reported as a VAChT mapping agent, and may be suitable for VAChT imaging studies.     

The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor

In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor.     

EGF promotes gastric mucosal restitution by activating Na(+)/H(+) exchange of epithelial cells

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na(+)/H(+) exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pH(i)) in SMC was measured fluorometrically, using 2',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pH(i) and enhanced restitution in the absence but not in the presence of serosal HCO. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na(+)/H(+) exchangers.