Aviglycine and propargylglycine inhibit conidial germination and mycelial growth of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>f. sp. <Emphasis Type="Italic">luffae</Emphasis>

Two inhibitors, aviglycine and propargylglycine, were tested for their ability to suppress methionine synthesis thus inhibit conidial germination and mycelial growth of Czapek-Dox liquid medium grown Fusarium oxysporum f. sp. luffae μM. The linear inhibition range for mycelial growth was about 7.6–762.9 μM. Although aviglycine did not completely inhibit both conidial germination and mycelial growth, it showed significant inhibitory effect at 1.5 μM. The inhibition range for propargylglycine against conidial germination and mycelial growth were from 0.08 to 8841 μM and from 0.8 to 884.1 μM, respectively. Propargylglycine inhibited conidial germination and mycelial growth at a concentration of 8841 μM. The EC₅₀ values of aviglycine were 1 μM for conidial growth and 122 μM for mycelial growth, and the EC₅₀ values of propargylglycine were 47.7 μM for conidial growth and 55.6 μM for mycelial growth. Supplement of methionine released inhibition of aviglycine or propargylglycine to conidial germination. In addition, a mixture of aviglycine (1.5 μM) and propargylglycine (8841 μM) showed additive inhibitive effect than applied alone on 10 isolates. From these results, both aviglycine and propargylglycine exhibited inhibitory activity, and suggest that they can provide potential tools to design novel fungicide against fungal pathogens.     

Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies

The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. Three human homologs, ABH1, ABH2 and ABH3, have been identified, and two of them, ABH2 and ABH3, were shown to have similar repair activities to E.coli AlkB. However, ABH1 did not show any repair activity. It was suggested that ABH3 prefers single-stranded DNA and RNA substrates, whereas AlkB and ABH2 can repair damage in both single- and double-stranded DNA. We employed a chemical cross-linking approach to probe the structure and substrate preferences of AlkB and its three human homologs. The putative active site iron ligands in these proteins were mutated to cysteine residues. These mutant proteins were used to cross-link to different DNA probes bearing thiol-tethered bases. Disulfide-linked protein–DNA complexes can be trapped and analyzed by SDS–PAGE. Our results show that ABH2 and ABH3 have structural and functional similarities to E.coli AlkB. ABH3 shows preference for the single-stranded DNA probe. ABH1 failed to cross-link to the probes tested. This protein, unlike other AlkB proteins, does not seem to interact with DNA in its E.coli expressed form.     

A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly

In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay.     

Estrogen ameliorates Nomega-nitro-L-arginine methyl ester-induced blood pressure increment in male spontaneously hypertensive rats: the role of cGMP

Estrogen (17beta-estradiol, or E2) reduces systolic blood pressure (SBP) increment and increases aortic cyclic guanosine monophosphate (cGMP) in male spontaneously hypertensive rats (SHRs). It is unknown, however, whether the E2-enhanced aortic cGMP is essential for the BP-lowering effect or not. Nomega-nitro-L-arginine-methyl ester (L-NAME), an L-arginine analogue and nitric oxide (NO) synthase inhibitor, significantly increases SBP and decreases aortic cGMP in male SHRs. We thus treated male SHRs with vehicle (corn oil) or E2 (s.c, 2 mg/kg/week) with or without L-NAME (20 mg/dl in the drinking water). SBP was measured weekly. Plasma nitrate/nitrite (NOx) concentrations and aortic cGMP levels were all measured at the end of the study. We found that SBP increment was significantly higher in L-NAME group, compared with the controls, and that E2 treatment reduced this L-NAME effect. Plasma 4NOx concentrations were not significantly different among different groups. Basal and acetylcholine-induced aortic cGMP, but not sodium nitroprusside-induced cGMP, were significantly lower in L-NAME group, compared with the controls. E2 co-administration did not modify L-NAME-induced aortic cGMP decrease. These data indicate that E2-induced BP-lowering effect in L-NAME treated male SHRs is not closely associated with the enhancement of vascular cGMP.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Computer-aided molecular design of novel glucosidase inhibitors for AIDS treatment

Since the onset of the AIDS epidemic, some 20 million people have died and the estimate is that today close to 40 million are living with type 1 human immunodeficiency virus (HIV)/AIDS. About 14 thousands people are infected worldwide daily with this disease. Still, only a few pharmaceuticals are available for AIDS chemotheraphy. Some pharmaceuticals act against the virus before the entrance of the HIV into the host cells. One of these targets is the glucosidase protein. This class of enzymes has been recently explored because the synthesis of viral glycoproteins depends on the activity of enzymes, such as glucosidase and transferase, for the elaboration of the polysaccharides. In this work we study several glucosidase inhibitors. The DFT method is used to compute atomic charges and the ligand/receptor interaction was simulated with docking software. Analysis of the interactions of the proposed pharmaceutical, a pseudodisaccharide, with the Thermotoga maritima 4-alpha-glucanotransferase in complex with modified acarbose, the scores from docking as well as the graphical superposition of all the ligands, suggest that our molecular designed pseudo-disaccharide may be a potent glucosidase inhibitor.