Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

Natural killer cells and innate immunity to protozoan pathogens

Natural killer (NK) cells are lymphoid cells that mediate significant cytotoxic activity and produce high levels of pro-inflammatory cytokines in response to infection. During viral infection, NK cell cytotoxicity and cytokine production is induced principally by monocyte-macrophage- and dendritic cell-derived cytokines but virally encoded ligands for NK cells are also beginning to be described. NK derived interferon-gamma (IFN-gamma) production is also essential for control of several protozoal infections including toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. The activation of NK cells by protozoan pathogens is also believed to be cytokine-mediated although some recent studies suggest that direct recognition of parasites by NK cells also occurs. Both indirect signalling via accessory cell-derived cytokines and direct signalling, presumably through NK receptors, are needed in order for human malaria parasites (Plasmodium falciparum) to optimally stimulate NK activity.     

An inhibitory Ig superfamily protein expressed by lymphocytes and APCs is also an early marker of thymocyte positive selection

Positive selection of developing thymocytes is associated with changes in cell function, at least in part caused by alterations in expression of cell surface proteins. Surprisingly, however, few such proteins have been identified. We have analyzed the pattern of gene expression during the early stages of murine thymocyte differentiation. These studies led to identification of a cell surface protein that is a useful marker of positive selection and is a likely regulator of mature lymphocyte and APC function. The protein is a member of the Ig superfamily and contains conserved tyrosine-based signaling motifs. The gene encoding this protein was independently isolated recently and termed B and T lymphocyte attenuator (Btla). We describe in this study anti-BTLA mAbs that demonstrate that the protein is expressed in the bone marrow and thymus on developing B and T cells, respectively. BTLA is also expressed by all mature lymphocytes, splenic macrophages, and mature, but not immature bone marrow-derived dendritic cells. Although mice deficient in BTLA do not show lymphocyte developmental defects, T cells from these animals are hyperresponsive to anti-CD3 Ab stimulation. Conversely, anti-BTLA Ab can inhibit T cell activation. These results implicate BTLA as a negative regulator of the activation and/or function of various hemopoietic cell types.     

Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.     

Intratumoral CC chemokine ligand 5 overexpression delays tumor growth and increases tumor cell infiltration

Chemokines participate in the antitumor immune response by regulating the movement and positioning of lymphocytes as well as effector functions and may thus be candidates for use in antitumor therapy. To test whether CCL5, a chemokine involved in the recruitment of a wide spectrum of immunocompetent cells, can control tumor growth, we forced its expression at mouse tumor sites. Tumor growth was reduced in mice with s.c. syngeneic CCL5-EL-4 compared with EL-4-injected mice, whereas both reduced tumor growth and incidence were observed in mice with OVA-expressing EG-7 transfected with CCL5 compared with EG-7-injected mice. Significant antitumor effects were observed soon after intratumoral injection of DNA plasmid coding for chimeric CCL5-Ig. Importantly, quantitative RT-PCR assays showed that the amount of CCL5 expression at the tumor site determined the effectiveness of the antitumor response, which was associated with infiltration of increased numbers of NK, CD4, and CD8 cells at the tumor site. This effect was lost in mice deficient for T/B lymphocytes (RAG-2 knockout) or for CCR5 (CCR5 knockout). Together, these data demonstrate the antitumor activity of intratumoral CCL5 overexpression, due to its recruitment of immunocompetent cells, and the potential usefulness of chimeric CCL5-Ig DNA as an agent in cancer therapy.     

Induction of disease by a molecularly cloned highly pathogenic simian immunodeficiency virus/human immunodeficiency virus chimera is multigenic

One of three full-length infectious molecular clones of SHIV(DH12R), designated SHIV(DH12R-CL-7) and obtained from productively infected rhesus monkey peripheral blood mononuclear cells, directed rapid and irreversible loss of CD4+ T cells within 3 weeks of its inoculation into Indian rhesus monkeys. Induction of complete CD4+ T-cell depletion by SHIV(DH12R-CL-7) was found to be dependent on inoculum size. The acquisition of this pathogenic phenotype was accompanied by the introduction of 42 amino acid substitutions into multiple genes of parental nonpathogenic SHIV(DH12). Transfer of the entire SHIV(DH12R-CL-7) env gene into the genetic background of nonpathogenic SHIV(DH12) failed to confer the rapid CD4+ T-lymphocyte-depleting syndrome; similarly, the substitution of gag plus pol sequences from SIV(smE543) for analogous SIV(mac239) genes in SHIV(DH12R-CL-7) attenuated the pathogenic phenotype. Amino acid changes affecting multiple viral genes are necessary, but insufficient by themselves, to confer the prototypically rapid and irreversible CD4+ T-cell-depleting phenotype exhibited by molecularly cloned SHIV(DH12R-CL-7).     

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.     

Cysteine as a Modulator Residue in the Active Site of Xenobiotic Reductase A: A Structural, Thermodynamic and Kinetic Study

Xenobiotic reductase A (XenA) from Pseudomonas putida 86 catalyzes the NADH/NADPH-dependent reduction of various substrates, including 2-cyclohexenone and 8-hydroxycoumarin. XenA is a member of the old yellow enzyme (OYE) family of flavoproteins and is structurally and functionally similar to other bacterial members of this enzyme class. A characteristic feature of XenA is the presence of a cysteine residue (Cys25) in the active site, where in most members of the OYE family a threonine residue is found that modulates the reduction potential of the FMN/FMNH^- couple. We investigated the role of Cys25 by studying two variants in which the residue has been exchanged for a serine and an alanine residue. While the exchange against alanine has a remarkably small effect on the reduction potential, the reactivity and the structure of XenA, the exchange against serine increases the reduction potential by +82 mV, increases the rate constant of the reductive half-reaction and decreases the rate constant in the oxidative half-reaction. We determined six crystal structures at high to true atomic resolution (d_min 1.03-1.80 Å) of the three XenA variants with and without the substrate coumarin bound in the active site. The atomic resolution structure of XenA in complex with coumarin reveals a compressed active site geometry in which the isoalloxazine ring is sandwiched between coumarin and the protein backbone. The structures further reveal that the conformation of the active site and substrate interactions are preserved in the two variants, indicating that the observed changes are due to local effects only. We propose that Cys25 and the residues in its place determine which of the two half-reactions is rate limiting, depending on the substrate couple. This might help to explain why the genome of Pseudomonas putida encodes multiple xenobiotic reductases containing either cysteine, threonine or alanine in the active site.     

Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.