Effect of liquid culture media on morphology,growth, propagule production,and pathogenic activity of the Hyphomycete,Metarhizium flavoviride

Two isolates of Metarhizium spp. were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M. flavoviride (or M. anisopliae var. acridum) strain IMI 330189, and Mf324 = M. flavoviride strain ARSEF324). Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects. Shake-flask experiments were carried out at 28 °C in the dark. Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity. Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules. Submerged propagule yields were higher withMf189 than with Mf324 in all seven media. While high concentrations of propagules (1.4 to 2.4 × 10⁸ propagules ml^-1 for MF189 and1.4 to 8.3 × 10⁷ propagules ml^-1 for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins–Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media). Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules. In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides. In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia. Infection potential of submerged propagules was assayed on Schistocerca gregaria. Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10⁷ propagules ml^-1. All seven media produced submerged propagules that were highly infectious for S. gregaria larvae. Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins–Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type 1 infection

Specific CD8 T-cell responses to human immunodeficiency virus type 1 (HIV-1) are induced in primary infection and make an important contribution to the control of early viral replication. The importance of neutralizing antibodies in containing primary viremia is questioned because they usually arise much later. Nevertheless antienvelope antibodies develop simultaneously with, or even before, peak viremia. We determined whether such antibodies might control viremia by complement-mediated inactivation (CMI). In each of seven patients studied, antibodies capable of CMI appeared at or shortly after the peak in viremia, concomitantly with detection of virus-specific T-cell responses. The CMI was effective on both autologous and heterologous HIV-1 isolates. Activation of the classical pathway and direct viral lysis were at least partly responsible. Since immunoglobulin G (IgG)-antibodies triggered the CMI, specific memory B cells could also be induced by vaccination. Thus, consideration should be given to vaccination strategies that induce IgG antibodies capable of CMI.     

A protein encoded by a member of the multicopy Ssty gene family located on the long arm of the mouse Y chromosome is expressed during sperm development

Multicopy Y-chromosomal genes in human and mouse have been postulated to play a role in spermatogenesis. The mouse Y long arm (Yq) carries hundreds of supposedly intronless copies of Ssty, for which no protein has hitherto been identified; mice lacking Yq are sterile with grossly abnormal sperm. We have now identified an Ssty-encoded protein (Ssty1) that is expressed in spermatids. The protein is absent from spermatids of mice that lack Yq, but is not reduced in mice with a two-thirds reduction of Ssty copies, implying that most do not produce this protein. Furthermore, no protein was produced by a strongly transcribed intronless Ssty transgene, raising doubts as to the protein-encoding potential of these intronless genes. We have now identified an intron-containing copy that is also present in multiple copies on Yq. One or more intron-containing copies are retained in the Ssty-deficient mice and may be the source of the Ssty1 protein.     

Identification of a subset of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains able to exploit an alternative coreceptor on untransformed human brain and lymphoid cells

The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4(+) transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4(-) astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Delta32/Delta32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.     

Evaluation of glycated glucagon-like peptide-1(7-36)amide in intestinal tissue of normal and diabetic animal models

Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully cross-reacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19% of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24--71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (<3 min) and bioactivity of the native peptide.     

Damage to secondary lymphoid follicles in AIDS-related persistent generalized lymphadenopathy, as revealed by the behaviour of dendritic reticulum cells possessing immunoreactive acid cysteine-proteinase inhibitor

It has been shown previously that dendritic reticulum cells (DRC) in human secondary lymphoid follicles possess an immunoreactive acid cysteine-proteinase inhibitor (ACPI). In the present study, lymph nodes from 12 patients with AIDS-related persistent generalized lymphadenopathy (PGL) were investigated in order to detect whether or not any alterations occur in ACPI-immunoreactive DRC in this disorder. In the majority of PGL cases, profound alterations were found, the main characteristics of which were erosion, partial or total disruption of lymphoid follicles. However, similar though much less marked alterations were also found in some control cases. It is concluded that this type of follicular damage is a common and characteristic feature in PGL. It is not specific to PGL, however, but represents rather a special type of reaction in lymphatic tissue. The advantage of ACPI immunohistology for demonstrating the DRC pattern is that it can be performed on routinely fixed and paraffin-embedded tissues.     

Steric and electronic requirements for muscarinic receptor-stimulated phosphoinositide turnover in the CNS in a series of arecoline bioisosteres.

A series of arecoline derivatives was utilized to assess steric and electronic effects important for activating muscarinic receptors in the CNS. Arecoline derivatives in which the methyl ester moiety was replaced by hexyloxy-1,2,5-oxadiazole (2b), hexyloxythiophene (3b) or hexyloxypyrazine (4b) were compared with the hexyloxy-1,2,5-thiadiazole compound (1b) (Hexyloxy-TZTP), known from previous work to be active as an M1/M3 partial agonist. MNDO calculations showed that the N-S bonds of the alkoxythiadiazole ring were highly polarized with the ability to form H-bonds to the N's. On the other hand, the smaller oxadiazole had lower polarities in the N-O bonds and reduced ability to form H-bonds, the thiophene was of comparable size to the thiadiazole and had large C-S bond polarities without the H-bond capability and the pyrazine had limited ability to form H-bonds. The compounds were compared with respect to their abilities to stimulate phosphoinositide (Pl) turnover in the hippocampus of the rat brain. 1b was more active than 2b-4b for stimulating the Pl turnover response. The data suggest that the ability to form H-bonds is an important factor for the ability of 1 to stimulate M1 muscarinic receptors in the CNS.     

Fish Viruses: Buffers and Methods for Plaquing Eight Agents Under Normal Atmosphere

A universal procedure was sought for plaque assay of eight fish viruses (bluegill myxovirus, channel catfish virus, eel virus, Egtved virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, lymphocystis virus, and the agent of spring viremia of carp (Rhabdovirus carpio), in dish cultures of various fish cells. Eagle minimal essential medium with sodium bicarbonate-CO(2) buffer (Earle's salt solution) was compared with minimal essential medium buffered principally with tris (hydroxymethyl)aminomethane or N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid at a pH or in the range of 7.6 to 8.0 depending upon temperature. Five fish cell lines collectively capable of replicating all fish viruses thus far isolated were tested and quantitatively found to grow comparably well in the three media. Two-phase (gel-liquid) media incorporating the various buffer systems allowed plaquing at 15 to 33 C either in partial pressures of CO(2) or in normal atmosphere, but greater efficiency and sensitivity were obtained with the organic buffers, and, overall, the best results were obtained with tris(hydroxymethyl)aminomethane. Epizootiological data, specific fish cell line response, and plaque morphology permit presumptive identification of most of the agents. At proper pH, use of organic buffers obviates the need for CO(2) incubators.     

Metabolism of phenanthrene by house fly CYP6D1 and dog liver cytochrome P450

Polycyclic aromatic hydrocarbons (PAHs) are a ubiquitous class of environmental contaminants. The compound phenanthrene is a model PAH. A novel fluorometric method for measuring phenanthrene metabolism in vitro was developed and verified with direct measurement of [14C]phenanthrene using dog liver microsomes. The fluorometric assay and direct measurement of [14C]phenanthrene metabolism were used to show that CYP6D1, a house fly cytochrome P450, is the major house fly P450 involved in phenanthrene metabolism. Phenanthrene was metabolized by microsomes from the LPR strain of house fly that overexpresses CYP6D1, but metabolism was not observed in the CS strain that has a lower level of CYP6D1. Furthermore, the majority of phenanthrene metabolism was inhibited by a CYP6D1-specific antibody. This study increases the number of known substrates of CYP6D1 and identifies polyaromatic hydrocarbons as potential substrates of CYP6D1. The utility of CYP6D1 as an agent in bioremediation and the utility of the new fluorometric assay for understanding PAH metabolism in insects and mammals are discussed.