Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus

We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway

Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK.     

Differential influence of increased polyol pathway on protein kinase C expressions between endoneurial and epineurial tissues in diabetic mice

To explore the relationship between polyol pathway and protein kinase C (PKC), we examined PKC activities and expressions of PKC isoforms separately in endoneurial and vessel-rich epineurial tissues in diabetic mice transgenic for human aldose reductase (Tg). Tg and littermate control mice (Lm) were made diabetic by streptozotocin at 8 weeks of age and treated orally with aldose reductase inhibitor (ARI) (fidarestat 3-5 mg/kg/day) or placebo for 12 weeks. At the end, compared with non-diabetic state, sorbitol contents were increased 6.4-fold in endoneurium and 5.1-fold in epineurium in diabetic Tg, whereas the increase was detected only in endoneurium in diabetic Lm. Endoneurial PKC activity was significantly reduced in diabetic Tg. By contrast, epineurial PKC activity was increased in both diabetic Lm and diabetic Tg and there was no significant difference between the two groups. These changes were all corrected by ARI treatment. Consistent with the changes of PKC activities, diabetic Tg showed decreased expression of PKC alpha in endoneurium, whereas there was an increased expression of PKC beta II in epineurium in both diabetic Tg and diabetic Lm. These findings suggest the presence of dichotomous metabolic pathway between neural and vascular tissues in the polyol-PKC-related pathogenesis of diabetic neuropathy.     

Ether-linked analogue of 2-arachidonoylglycerol (noladin ether) was not detected in the brains of various mammalian species

2-Eicosa-5',8',11',14'-tetraenylglycerol (2-AG ether, HU310, noladin ether) is a metabolically stable ether-linked analogue of 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand. 2-AG ether has been used as a valuable experimental tool by a number of investigators. Recently, several groups reported that 2-AG ether is present in mammalian brains. We examined in detail whether 2-AG ether actually exists in the brains of various mammalian species. We found that 2-AG ether is not present, at least in an appreciable amount, in the rat brain by gas chromatography-mass spectrometry analysis and fluorometric high performance liquid chromatography analysis. The level of 2-AG ether in the rat brain was below 0.2 pmol/g brain, if at all present. Similar results were obtained for the mouse brain, hamster brain, guinea-pig brain and pig brain. The fact that 2-AG ether was not detected in the brains of various mammalian species is consistent with the fact that an ether bond is formed through enzymatic replacement of the fatty acyl moiety of 1-acyl dihydroxyacetone phosphate by a fatty alcohol, the resultant 1-O-alkyl dihydroxyacetone phosphate being a common intermediate of the biosynthesis of ether-linked lipids in mammalian tissues. It is rather questionable whether 2-AG ether is present in appreciable amounts in the brain and acts as an 'endogenous' cannabinoid receptor ligand.     

Isolation and molecular characterization of a nelfinavir (NFV)-resistant human immunodeficiency virus type 1 that exhibits NFV-dependent enhancement of replication

During the use of a phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay in a large set of clinical virus isolates, we found a unique variant (CL-4) that exhibited a high level of nelfinavir (NFV) resistance and rather enhanced replication under subinhibitory concentrations of NFV (0.001 to 0.1 micro M). Comparison of gag-pol sequences of the CL-4 variant and its predecessor virus isolates showed a stepwise accumulation of a total of 19 amino acid substitutions in protease (PR) and Gag p17 during 32-month NFV-containing antiretroviral therapy, while other Gag regions including the cleavage sites of the p55 precursor remained highly conserved. To understand the relationship between the genetic and phenotypic changes in CL-4, we constructed chimeric viruses using pNL4-3, replacing the PR, p24PR, or p17PR gene segment of CL-4 or its predecessor. A series of tissue culture infections with the chimeras in the absence or presence of increasing concentrations of NFV demonstrated that only the p17PR segment of CL-4 could confer the NFV-dependent replication enhancement phenotype on NL4-3. Our data suggest a novel adaptation mechanism of HIV-1 to NFV, in which coevolution of Gag and PR genes generates a variant that replicates more efficiently in the cellular environment in the presence of NFV than without the drug.     

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.