Redressing the sex imbalance in knowledge of vector biology

The recent development of transgenic mosquitoes that are resistant to infection by the Plasmodium malarial parasite is a promising new tool in the fight against malaria. However, results of large-scale field releases of alternatively modified mosquitoes carried out during the 1970s and 1980s suggest that this approach could be difficult to implement in the field. These past attempts to control mosquito populations largely floundered as a result of our insufficient understanding of the behavioural ecology of released males. In spite of this, contemporary research on genetic control strategies has concentrated predominantly on molecular aspects, with little progress being made toward resolving key ecological uncertainties, male mosquito ecology being the most important. Here, we review the state of knowledge of male mosquito ecology, and highlight priorities for further research. Case studies of two crop pests, the Mediterranean fruit fly and melon fly, are given as examples of how knowledge of male ecology facilitates successful control in other species. Unless similar information becomes available for mosquitoes, any future genetic control strategy will risk failure.     

Viral protein VP4 is a target of human antibodies enhancing coxsackievirus B4- and B3-induced synthesis of alpha interferon

Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-alpha) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-alpha were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56 degrees C from CVB4E2 (VP4(CVB4)) and CVB3 (VP4(CVB3)) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis. There was no cross-reaction between VP4(CVB4) and VP4(CVB3) in the inhibiting effect. IFN-alpha levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4(CVB4)). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-alpha levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis by PBMC.     

Biocatalysed synthesis of beta-O-glucosides from 9-fluorenon-2-carbohydroxyesters. Part 3: IFN-inducing and anti-HSV-2 properties

In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the 'retaining' beta-glycosidase from the archaeon Sulfolobus solfataricus (Ssbeta-Gly). Both aglycones (1-6) and corresponding beta-glucosides (beta-glu 1-beta-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds beta-glu 5, beta-glu 6 and beta-glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-alpha/beta. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.     

Strategies for gathering structural information on unknown peaks in the GC/MS analysis of Corynebacterium glutamicum cell extracts

A comprehensive analysis of low molecular weight compounds in biological samples by the hyphenated method of GC/MS in general detects a large number of peaks which can not be identified by searching of commercially available mass spectral libraries. Therefore, more information is required for a successful identification of these compounds. Some structural features like molecular weight and number of derivatization groups present in the molecule can be determined by variation of the derivatization prior to GC/MS. The use of deuterated derivatizing reagents and the newly developed N-methyl-N-ethyldimethylsilyl-trifluoracetamide for this purpose is described. The knowledge of these structural properties alone undoubtedly will not lead to the structure elucidation of novel metabolites. However, it may be helpful in identifying derivatives of known metabolites or artifacts. Thus, by use of the molecular weight as search criterion it is possible to find plausible candidates in metabolic pathway databases. It is shown that the application of this method to a hydrophilic extract of C. glutamicum lead to the identification of 31 in previous analyses unidentified peaks, in their majority representing minor derivatives of common metabolites.     

Glycosylphosphatidylinositol-anchored fungal polysaccharide in Aspergillus fumigatus

Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C18-phytosphingosine and monohydroxylated lignoceric acid (2OH-C(24:0) fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN. The galactomannan polymer is linked to the GPI structure through the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.     

The epidermal barrier function is dependent on the serine protease CAP1/Prss8

Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (approximately 600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival.     

Membrane topology of loop 13-14 of the Na+/glucose cotransporter (SGLT1): a SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na+/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES(-) and MTSET(+) had no effect on either cotransport or Na+ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K(i)(Pz) and K(m)(alphaMG) for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K(i)(Pz) values than wtSGLT1 with minimal changes in K(m)((alpha)MG), the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.