MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 5: Carbon-substituted analogues at the C-2 position

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with carbon-linked substituents, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Palladium-catalyzed cross-coupling methods were applied for the incorporation of aliphatic and aromatic substituents.     

Decreased exploratory activity in a mouse model of 15q duplication syndrome; implications for disturbance of serotonin signaling

Autism spectrum disorders (ASDs) have garnered significant attention as an important grouping of developmental brain disorders. Recent genomic studies have revealed that inherited or de novo copy number variations (CNVs) are significantly involved in the pathophysiology of ASDs. In a previous report from our laboratory, we generated mice with CNVs as a model of ASDs, with a duplicated mouse chromosome 7C that is orthologous to human chromosome 15q11-13. Behavioral analyses revealed paternally duplicated (patDp/+) mice displayed abnormal behaviors resembling the symptoms of ASDs. In the present study, we extended these findings by performing various behavioral tests with C57BL/6J patDp/+ mice, and comprehensively measuring brain monoamine levels with ex vivo high performance liquid chromatography. Compared with wild-type controls, patDp/+ mice exhibited decreased locomotor and exploratory activities in the open field test, Y-maze test, and fear-conditioning test. Furthermore, their decreased activity levels overcame increased appetite induced by 24 hours of food deprivation in the novelty suppressed feeding test. Serotonin levels in several brain regions of adult patDp/+ mice were lower than those of wild-type control, with no concurrent changes in brain levels of dopamine or norepinephrine. Moreover, analysis of monoamines in postnatal developmental stages demonstrated reduced brain levels of serotonin in young patDp/+ mice. These findings suggest that a disrupted brain serotonergic system, especially during postnatal development, may generate the phenotypes of patDp/+ mice.     

Blocking of human immunodeficiency virus type-1 virion autolysis by autologous p2(gag) peptide

Our previous study suggested that the p2(gag) peptide, AEAMSQVTNTATIM, inhibits human immunodeficiency virus type 1 (HIV-1) protease (PR) activity in vitro. In this study, Ala substitutions (Met4Ala and Thr8Ala) and deletion of amino acid Asn9 within the nona p2(gag) peptide (AEAMSQVTN) were found to decrease the inhibitory effect on HIV-1 PR activity. Furthermore, treatment of PMA-activated latently infected T lymphocytes, ACH-2 cells, with the p2(gag) peptide (100 and 250 micro M) resulted in a decrease in the amount of p24(gag )in the resultant viral lysates derived from the cell-free supernatant. In addition, the HIV-1-Tat-p2(gag) fusion peptide was synthesized to effectively deliver the p2(gag) peptide into the cells. The fusion peptide was incorporated into chronically infected T lymphocytes, CEM/LAV-1 cells, as detected on indirect immunofluorescence analysis using anti-p2(gag) peptide monoclonal antibodies, which recognize the nona peptide (AEAMSQVTN) derived from the N-terminus of the p2(gag) peptide, and cleaved by HIV-1 PR in vitro. Treatment of CEM/LAV-1 cells with the fusion peptide also resulted in a decrease in the amount of p24(gag )in the resultant viral lysate derived from the cell-free supernatant. Taken together, these data suggest that the p2(gag) peptide consequently blocks the autolysis of HIV-1 virions for the conservation of viral species.     

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.     

Betagamma-dehydrocurvularin and related compounds as nematicides of Pratylenchus penetrans from the fungus Aspergillus sp

The new nematicidal compound, betagamma-dehydrocurvularin (1), together with three known compounds, alphabeta-dehydrocurvularin (2), 8-beta-hydroxy-7-oxocurvularin (3) and 7-oxocurvularin (4), were isolated from the culture filtrate and mycelial mats of Aspergillus sp. The structures of 1-4 were established by spectroscopic methods including 2D NMR. The biological activities of 1-4 were examined by bioassays with root-lesion nematodes, and lettuce and rice seedlings.     

A novel cyclic peptide immunization strategy for preventing HIV-1/AIDS infection and progression

A novel synthetic peptide immunogen targeting the human immunodeficiency virus type-1 (HIV-1) coreceptor CXCR4 was evaluated for its capacity to induce CXCR4-specific antibodies with anti-HIV-1 activity in BALB/c mice and cynomolgus monkeys. A cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4 (cDDX4) was prepared in which Gly-Asp, as the dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (linear DDX4, Asn176 to Ile185) derived from the undecapeptidyl arch (UPA; Asn176 to Cys186) of extracellular loop 2 (ECL-2) in CXCR4. Immunization of BALB/c mice with cDDX4 conjugated with a multiple-antigen peptide (cDDX4-MAP) induced conformational epitope-specific antibodies, and monoclonal antibody IA2-F9 reacted with cDDX4, but not with linear DDX4, as determined by real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CXCR4 but not with cells expressing the other HIV coreceptor, CCR5. Furthermore, the antibody inhibited the replication of HIV-1 X4 virus (using CXCR4), as shown by an infection assay using both MAGIC-5 cells and MT4 cells, but not that of HIV-1 R5 virus (using CCR5). The antibody weakly interfered with chemotaxis induced by stromal cell-derived factor-1 alpha in THP-1 cells or moderately inhibited the chemotaxis of Molt4#8 cells under the same conditions. In addition, immunization of cynomolgus monkeys also induced cDDX4-specific antibodies with anti-HIV activity. Taken together, these results indicate that cDDX4 conjugated with a multi-antigen peptide induces the conformational epitope-specific antibodies to the undecapeptidyl arch of CXCR4 may be a novel candidate immunogen for preventing disease progression in HIV-1-infected individuals.     

A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450

We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.     

The identification of CVP1 reveals a role for sterols in vascular patterning

Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.