Antioxidant Enzyme Level Response to Prooxidant Allelochemicals in Larvae of the Southern Armyworm Moth, Spodoptera Eridania

Larvae of the southern armyworm, Spodoplera eridania, are highly polyphagous feeders which frequently encounter and feed upon plants containing high levels of prooxidant allelochemicals. While ingestion of moderate quantities of prooxidants can be tolerated by these larvae, ingestion of larger quantities can result in toxicity. Studies were conducted to assess the role of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) in the protection of S. eridania against redox active prooxidant plant allelochemicals. Dietary exposure of mid-fifth-instar larvae to either quercetin (a flavonoid) or xanthotoxin (a photoactive furanocoumarin), which generate superoxide radical, and singlet oxygen, repsectively, resulted in an increase in SOD levels. CAT levels increased in all groups of S. eridania including control insects. This may have been due to the sudden exposure to food following an extended fast of 18 h (to insure that larvae would not reject the diet because of the prooxidants' bitter taste) with an eventual lowering of CAT values with time. GR activities did not significantly change except for a slight inhibition at the highest prooxidant concentrations used at 12-h post-ingestion. The data from these studies suggest that SOD responds to prooxidant challenges in these insects and together with CAT and GR contributes to the insect's defense against potentially toxic prooxidant compounds.     

Detection of type-specific antibody to lymphogranuloma venereum serotypes ofChlamydia trachomatis with a microneutralization test

Ten-day monospecific mouse antisera prepared against serotypes L1, L2, and L3 ofChlamydia trachomatis were titrated against homologous and heterologous antigens in the microimmunofluorescence (micro-IF) test and by a new microneutralization method. While broad crossreactivity between serotypes was detected in the micro-IF test, the same antisera demonstrated serotype specific neutralizing activity. The microneutralization test could be used for serotyping new chlamydial isolates when equivocal results have been obtained by the microimmunofluorescence method.     

Encounters of old foes on a new battle ground for an invasive tree, Albizia julibrissin Durazz (Fabaceae)

Studies of invasive species often demonstrate that exotic or invasive plants have reduced levels of predation when first introduced into novel geographic areas. In some systems, native predators may subsequently be introduced and the plant-predator association is re-established. In this study, we analyzed a recent reassociation between the introduced tree, Albizia julibrissin, and its native seed predator, Bruchidius terrenus, which has also been introduced, in the vicinity of Athens, GA, USA. We documented a rapid increase in the levels of bruchid beetle predation on seeds of A. julibrissin from 2001 to 2002 and substantial predation in the following 5 years. From a six-year survey, we found that relative susceptibility to beetle predation among individual trees remained consistent over time. Furthermore, we found that phenology had significant effects on beetle predation on fruits and seeds, with fruits developed from early flowers having higher levels of beetle attacks than ones developed from later flowers in the season. Finally, seeds and fruits attacked by beetles were more likely to be attacked by a Fusarium fungus, probably due to coinfestation of the two predators. Host-predator reassociation that causes substantial loss of fertility or shows a relationship with reproductive phenology may have population level demographic effects or evolutionary consequences and deserve further research attention.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Biosynthesis of Vitamin B6 by a Yeast Mutant

The gradient-plate technique was employed to isolate mutants of Saccharomyces marxianus (NRRL-Y-1550) which, when grown in a synthetic culture medium, excreted about 2 mug/ml of vitamin B(6) as ascertained by microbiological assay. The major component that possessed vitamin B(6) activity was isolated by ion-exchange column chromatography and identified as pyridoxol by ultraviolet and fluorescence spectroscopy, as well as by paper chromatography and various chemical tests. Pyridoxal was also identified as one of the excreted compounds. Two other compounds that possessed vitamin B(6) activity were excreted in smaller quantities in the growth medium and have not yet been identified; they are not phosphates of vitamin B(6). The amount of vitamin B(6) excreted was not increased when the mutant was grown in the presence of various oxidation products of this vitamin. The methods and results reported here may be helpful in future studies on the biosynthesis of vitamin B(6).     

Prevalence and Serological Diagnosis of Relapse in Paracoccidioidomycosis Patients

A review of 400 clinical records of paracoccidioidomycosis (PCM) patients, 93 with the acute/subacute (AF) and 307 with the chronic form (CF), attended from 1977 to 2011, selected as to the schedule of release for study by the Office of Medical Records at the University Hospital of the Faculdade de Medicina de Botucatu – São Paulo State University – UNESP, was performed to detect cases in relapse. The control of cure was performed by clinical and serological evaluation using the double agar gel immunodiffusion test (DID). In the diagnosis of relapse, DID, enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay (IBgp70 and IBgp43) were evaluated. Out of 400 patients, 21 (5.2%) went through relapse, 18 of them were male and 3 were female, 6∶1 male/female ratio. Out of the 21 patients in relapse, 15 (4.8%) showed the CF, and 6 (6.4%) the AF (p>0.05). The sensitivity of DID and ELISA before treatment was the same (76.1%). DID presented higher sensitivity in pre-treatment (80%) than at relapse (45%; p = 0.017), while ELISA showed the same sensitivity (80% vs 65%; p = 0.125). The serological methods for identifying PCM patients in relapse showed low rates of sensitivity, from 12.5% in IBgp70 to 65.0% in IBgp43 identification and 68.8% in ELISA. The sensitivity of ELISA in diagnosing PCM relapse showed a strong tendency to be higher than DID (p = 0.06) and is equal to IBgp43 (p = 0.11). In sum, prevalence of relapse was not high in PCM patients whose treatment duration was based on immunological parameters. However, the used methods for serological diagnosis present low sensitivity. While more accurate serological methods are not available, we pay special attention to the mycological and histopathological diagnosis of PCM relapse. Hence, direct mycological, cytopathological, and histopathological examinations and isolation in culture for P. brasiliensis must be appropriately and routinely performed when the hypothesis of relapse is considered.     

Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly,Papilio polyxenes

The black swallowtail butterfly, Papilio polyxenes, larvae are specialized feeders of pro-oxidant rich plants of Apiaceae and Rutaceae. An important defense against toxic forms of oxygen species generated by ingestion of the pro-oxidants, are the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), GSH-dependent glutathione peroxidases (selenium-dependent glutathione peroxidase [GPOX] and peroxidase activity of selenium-independent glutathione-S-transferase [GT_px]), and glutathione reductase (GR). The subcellular distribution of these enzymes in black swallowtail larvae was investigated and was found to resemble the patterns described for larvae of two other lepidopteran species: the southern armyworm, Spodoptera eridania, and the cabbage looper, Trichoplusia ni. The confinement of SOD in the cytosol and mitochondria was typically eukaryotic, but the relative proportion (1:1) was markedly different from the mammalian pattern (4:1; cytosol:mitochondria). The most obvious difference between the black swallowtail and other lepidoptera as a group, and mammalian species, is in very wide intracellular distributions of CAT, GTpx, and GR in insect species. Insects possess very low levels of a GPOX-like activity which reduces both H₂O₂ and organic peroxides. Consequently, insects have elaborate activities with a wide subcellular distribution of both CAT which decomposes H₂O₂, and GTpx which decomposes organic peroxides. The reduction of peroxides is dependent on GSH, which in this process is oxidized to GSSG. GR which reduces GSSG to GSH is also of wide subcellular distribution, analogous to the distribution pattern of GTpx.