Parasitic effect of Opius concolor (Spzl) (Hymenoptera, Braconidae) on an intermediate host Ceratitis capitata Wied (Diptera, Trypetidae)

The parasitic incidence of Opius concolor (Hymenoptera, Braconidae) on a replacement host, Ceratitis capitata (Diptera, Trypetidae) was studied using polyacrylamide gel electrophoresis and immunoelectrophoresis methods. A noticeable modification of C. capitata proteins was observed when parasited by O. concolor. But the most important phenomenon is the finding of common antigens between the host and the parasite. These results are discussed with regard to trophic and parasitic behaviour of the parasite.     

Electrical conduction through nerve and DNA

The aim of the present study was to analyse electric resistivity at different ambient temperatures between 300 to 20K in the frog sciatic nerve and salmon sperm DNA. When the electrical contacts were leaned just into the sciatic nerve, an increase of the sciatic nerve resistivity was observed for 240 K < T < 300 K and a rise of electrical conductivity was apparent below 240 K. This dependence is generally associated with a semiconductor behaviour. Once the sciatic nerve temperature was driven below 250K, the resistivity abruptly decreased and then at temperatures lower than 234 K, it remained constant and close to one tenth of its ambient temperature value. By contrast, when the electrical contacts were leaned into Salmon sperm DNA, the resistivity remained constant between 300K to 20K, showing a high electrical stability at low temperature. Thus, we report the existence of a new form of electric conductivity in the sciatic nerve at low ambient temperature, which in turn has many electric similarities with inorganic or organic superconductors, whereas temperature failed to alter DNA electrical properties until 20K.     

A rapid staining technique for the detection of the initiation of germination of bacterial spores

AIMS: We propose to apply the Wirtz-Conklin staining technique to evaluate spore germination. METHODS AND RESULTS: Spores at different stages of germination were stained with modified spore stain (Wirtz-Conklin) and evaluated for staining properties. Bacillus spores suspended in deionized water, which does not support germination, stained greenish-blue. Spores suspended in germination enhancers that did not form bacilli stained pink, indicating the initiation of germination. Spores suspended in culture media, which promotes bacterial outgrowth, formed bacilli and were also stained pink. CONCLUSIONS: Modified spore stain (Wirtz-Conklin) was found to be useful to detect the initiation of spore germination as early as 30 min following incubation in a germination environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple staining procedure is useful in detecting the initiation of germination of bacterial spores.     

Leishmania infantum species-specific kDNA probes: isolation and evaluation

The study refers to the isolation of specific DNA probes to the parasite species Leishmania (L) infantum according to different strategies using recombinant minicircles isolated from L. infantum kinetoplast DNAs. A first probe was identified following a classical procedure. One mini-circle selected for strong reactivity to L. infantum total DNA was used to identify specific subfragments to this species among which the 95bp fragment, 3B8HaeIII-2 was selected. For the obtention of the second probe, a strategy based on sequential screenings for specificity and sensitivity was applied. This allowed identification of a set of minicircles showing an increased specificity to L. infantum as compared to other species, and an increased sensitivity of reaction as compared to the other minicircles. Subclonings and screenings allowed a final selection of a 137bp-minicircle fragment: 3E9HaeIII-12. Reactivities of the 2 probes were assessed on a panel of total DNAs and promastigotes from 74 isolates pertaining to 9 species encountered in the Old World. Parasites isolated in Tunisia from different foci, different hosts after different transmission seasons were included. Hybridizations have shown the exquisite specificity of these probes to L. infantum in this country. Probe 3E9HaeIII-12 was found to be the more sensitive where down to 10 ng of total DNA and 10(3) promastigotes could be detected. From this study and as compared to data provided in the literature, the second procedure allowed at least 10-fold increase in sensitivity.     

The fungicidal activity of novel nanoemulsion (X8W60PC) against clinically important yeast and filamentous fungi

Surfactant nanoemulsions are water in oil preparations that proved to have a broad spectrum biocidal activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria, spores and enveloped viruses. These preparations are non-toxic to the skin, mucous membrane and gastrointestinal tissues at biocidal concentrations. In this study, 0.1% of the nanoemulsion designated X8W₆₀PC has shown fungicidal activity against yeast including Candida albicans and C. tropicalis in 15 minutes. C. tropicalis was more sensitive than C. albicans, which required a longer time or a higher concentration of the nanoemulsion to achieve killing. Neutral to slightly alkaline pH was more effective in killing the yeast cells than acidic pH. Using the minimum inhibitory concentration assay, 0.08% of the nanoemulsion was inhibitory to C. albicans, and parapsilosis and filamentous fungi including Microsporum gypseum,Trichophyton mentagrophytes,Trichophyton rubrum,Aspergillus fumigatus andFusarium oxysporum.None of the individual ingredients was as effective a fungicidal as the nanoemulsion at equivalent concentration. This shows that the nanoemulsion structure is an important factor in the anti-fungal activity. The X8W₆₀PC has great potential as a topical anti-fungal agent and further investigation into the mechanism of fungicidal action is warranted.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing

Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.     

Toxoplasma gondii: Ultrastructure study of the entry of tachyzoites into mammalian cells

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5–15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48–72 h.     

A novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without Th2 associated vaccine-enhanced disease

Background

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960''s led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations.

Principal Findings

In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines.

Conclusions

These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.     

Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.