Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.     

High dose Losartan and ACE gene polymorphism in IgA nephritis

Background/aims Several studies have reported varying results of the influence of ACE gene on ACEI/ARB therapy. The efficacy of high dose ARB and its influence on ACE gene have not been explored. This is a 6 year randomised trial in IgA nephritis comparing high dose ARB (Losartan 200 mg/day) with normal dose ARB (Losartan 100 mg/day), normal dose ACEI (20 mg/day) and low dose ACEI (10 mg/day). Results Patients on high dose ARB had significantly lower proteinuria, 1.0 ± 0.8 gm/day compared to 1.7 ± 1.0 g/day in the other groups (P = 0.0005). The loss in eGFR was 0.7 ml min^?1year^?1 for high dose ARB compared to 3.2–3.5 ml min^?1year^?1 for the other three groups (P = 0.0005). There were more patients on high dose ARB with improvement in eGFR compared to other three groups (P < 0.001). Comparing patients with the three ACE genotypes DD, ID and II, all three groups responded well to therapy with decrease in proteinuria (P < 0.002). Only those on low dose ACEI (10 mg/day) with the I allele had increased in ESRF (P = 0.037). Conclusion High dose ARB is more efficacious in reducing proteinuria and preserving renal function when compared with normal dose ARB and ACEI, and also obviates the genomic influence of ACE gene polymorphism on renal survival.     

Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten–protein conjugate for antibody development

Polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline (PS-IIDQ), a polymer-supported covalent coupling reagent, was successfully employed for the first time in the bioconjugation of an example hapten (phytanic acid derivative) to a carrier protein (bovine serum albumin (BSA)) within the context of immunogen preparation for antibody development. The ability of the prepared example phytanic acid derivative–BSA conjugate to bind an anti-phytanic acid antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA).     

ExCyto PCR Amplification

Background

ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme.

Results

Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated.

Conclusions

ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research.     

Evolutionary relationships and polymorphisms among V κ 10 genes from inbred and wild-derived inbred mice

The V kappa 10 family in BALB/c mice is composed of three members, two of which are utilized in a variety of immune responses. We previously demonstrated that the product of the third gene, V kappa 10C, has never been detected as part of a functional antibody and productive rearrangements are selectively lost during B-cell development. Here we analyzed germline V kappa 10 genes from inbred and wild-derived mice by RFLP and sequencing in order to determine the origin of the V kappa 10C gene, as well as to examine the evolutionary relationships of V kappa 10 genes. Our results demonstrated that the V kappa 10 family is highly conserved across Mus species and subspecies, but that V kappa 10C is rare, being found in only inbred mice of V kappa 10 allelic group b and two of six M. m. domesticus isolates. It was not found in other M. musculus subspecies or M. spretus. V kappa 10A and V kappa 10B were found in all strains, with the exception of one M. m. domesticus isolate, which had only V kappa 10B genes. Overall, V kappa 10A sequences were more highly conserved than V kappa 10B, indicating that different selective pressures may be operating on these genes. The two V kappa 10C sequences from M. m. domesticus were 100% identical to that found in inbred mice. V kappa 10C is more closely related to V kappa 10B than to V kappa 10A and our data suggest that it is a recent duplication of the V kappa 10B gene.     

Gamma-secretase: substrates and inhibitors

The amyloid β-protein (Aβ) deposited in Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a secreted proteolytic product of the amyloid β-protein precursor (APP). Generation of Aβ from the APP requires two sequential proteolytic events, β-secretase cleavage to generate the amino terminus, followed by γ-secretase cleavage to generate the carboxyl terminus. Because this process is a central event in the pathogenesis of AD, γ-secretase is believed to be an excellent therapeutic target. γ-Secretase activity has been demonstrated to be membrane-associated, with the cleavage site primarily determined by the location of the substrate with respect to the membrane. It has also been shown that this unusual proteolytic activity not only occurs for APP, but also for proteins involved in morphogenic processes or cell proliferation and differentiation such as Notch and ErbB4. Thus far, all γ-secretase substrates are involved in some form of nuclear signaling. These recent findings have important implications for the development of pharmacological interventions that target γ-secretase.     

Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae

Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.     

CYTONUCLEAR THEORY FOR HAPLODIPLOID SPECIES AND X-LINKED GENES. II. STEPPING-STONE MODELS OF GENE FLOW AND APPLICATION TO A FIRE ANT HYBRID ZONE

We develop cytonuclear, hybrid zone models for haplodiploid species or X-linked genes in diploid species using a stepping-stone framework of migration, in which migration rates vary with both direction and sex. The equilibrium clines for the allele frequencies, cytonuclear disequilibria, and frequencies of pure parental types are examined for species with diagnostic markers, under four important migration schemes: uniform migration of both sexes in both directions, greater migration of both sexes from one direction, greater migration of females, and greater migration of males. Of the three cytonuclear variables examined, the allele frequency clines are the most informative in differentiating among the various migration patterns. The cytonuclear disequilibria and the frequency of the pure parental types tend to be useful only in revealing directional asymmetries in migration. The extent of hybrid zone subdivision has quantitative but not qualitative effects on the distribution of cytonuclear variables, in that the allele frequency clines become more gradual, the cytonuclear disequilibria decrease in magnitude, and the frequencies of pure parentals decline with increasing subpopulation number. Also, the only major difference between the X-linked and haplodiploid frameworks is that a higher frequency of pure parentals is found when considering haplodiploids, in which male production does not require mating. The final important theoretical result is that censusing after migration yields greater disequilibria and parental frequencies than censusing after mating. We analyzed cytonuclear data from two transects from a naturally occurring hybrid zone between two haplodiploid fire ant species, Solenopsis invicta and S. richteri, using our stepping-stone framework. The frequency of S. invicta mtDNA exceeds the frequency of the S. invicta nuclear markers through much of this hybrid zone, indicating that sex differences in migration or selection may be occurring. Maximum-likelihood estimates for the migration rates are very high, due to an unexpectedly large number of pure parental types in the hybrid zone, and differ substantially between the two transects. Overall, our model does not provide a good fit, in part because the S. invicta–S. richteri hybrid zone has not yet reached equilibrium.     

Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influence of diffusion in biological reaction systems. The broad substrate specificity and hydrolytic efficiency of OPH coupled with the ability to genetically engineer the enzyme for specific target OP neurotoxins enhance the suitability of OPH-based technologies for detoxification of these compounds. Cryoimmobilization provides a suitable vehicle as a cost-effective, efficient technology for bioremediation of environmental media contaminated with OP compounds.