Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae

The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.     

Association of plasma viral RNA load with prognosis in cats naturally infected with feline immunodeficiency virus

We measured the quantity of plasma feline immunodeficiency virus (FIV) RNA using a real-time sequence detecting system. Plasma viral RNA load was shown to correlate with the clinical stage, survival time, and disease progression in naturally FIV-infected cats. The present study indicates that the plasma viral RNA load can be used as a clinical marker representing the impairment of the immune system and predicting the clinical outcome in FIV-infected cats.     

Termite ecology in a dry evergreen forest in Thailand in terms of stable (δ 13C and δ 15N) and radio (14C, 137Cs and 210Pb) isotopes

Stable (¹³C and ¹⁵N) and radio- (¹⁴C, ¹³⁷Cs and ²¹⁰Pb) isotopes were determined for termites that have been sampled from a dry evergreen forest in Thailand. A wood-feeding termite, Microcerotermes crassus, was separated from soil-feeders: Termes propinquus, Termes comis and Dicuspiditermes makhamensis by ¹³C and ¹⁵N values. The Termes group in Thailand had less diverse values in ¹³C and ¹⁵N than those in Australia, where the feeding habits of the Termes group are more diverse. Other soil-feeding termites produced similar ¹³C values, but a larger range in ¹⁵N values. ¹⁴C-percent modern carbon (pMC) values suggest that the soil-feeding termites used younger carbon than the wood-feeding termites, and this was consistent with the termites from Cameroon, central Africa. Values of ¹³C and ¹⁴C-pMC indicate that surface soil was used by a soil-feeding termite, D. makhamensis, in making the nest mounds, and deeper soil (10–30 cm) by a fungus-growing termite, Macrotermes carbonarius. ²¹⁰Pb and ¹³⁷Cs were scarcely incorporated into the termites, although ²¹⁴Pb was recovered from the workers. The results suggest that stable- and radioisotopes are useful in the study of detritivorous animals, organic matter decomposition and ecosystem engineering.Takuya Abe - deceased.     

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety,and oral analgesic efficacy in rat pain models

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.     

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.     

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.     

Changes in lignin content of leaf litters during mulching

Alkaline nitrobenzene oxidation, ozonation and methoxyl content determinations were applied to decomposing leaf litter of Ginkgo biloba L., Cinnamomum camphora sieb., Zelkova serrata Makino and Firmiana simplex W. F. Wight, respectively, during mulching to investigate the properties and estimate changes in lignin composition and content. Since the Klason lignin residue originated from components highly resistant to degradation by acid, the methoxyl content of Klason residue was used to estimate the lignin content of leaf litter. Quantitative analysis of presumed lignin-derived fragments, by use of alkaline nitrobenzene oxidation and ozonation methods, suggested that the estimated lignin content approximates that of the real lignin content of leaves, which is greatly overestimated by the Klason procedure. The estimated lignin contents ranged from 3.9 to 10.0% while the Klason lignan residue varied from 37.1 to 46.7% in un-mulched leaf litter. The absolute amounts of the measured lignin somewhat decreased during mulching, while the structure of lignin remaining in leaf litters after mulching was considered not to be very different from its original structure.     

Proteomic approach to apoptotic thymus maturation

Apoptosis is an essential process for selection of T lymphocytes specific for foreign antigen in the process of mammalian thymus maturation. Proteomics, a comprehensive study of proteins expressed in a cell, will facilitate the systematic analysis of protein molecules related to such a complicated biological system. Protein expression profiles including information about protein signatures, localization and their quantitative changes with extracellular stimulations are extremely useful to construct intracellular pathway models resulting in the apoptotic cell death.     

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.