The hisC1 gene, encoding aromatic amino acid aminotransferase-1 in Azospirillum brasilense Sp7, expressed in wheat

Background and aims

Production of indole-3-acetic acid (IAA) by Azospirillum brasilense is one of the most important mechanisms underlying the beneficial effects observed in plants after inoculation with this bacterium. This study determined the contribution of the hisC1 gene, which encodes aromatic amino acid aminotransferase-1 (AAT1), to IAA production, and analyzed its expression in the free-living state and in association with the roots of wheat.

Methods

We determined production of IAA and AAT activity in the mutant hisC::gusA-sm ^R . To study the expression of hisC1, a chromosomal gene fusion was analyzed by following β-glucuronidase (GUS) activity in vitro, in the presence of root exudates, and in association with roots.

Results

IAA production in the hisC mutant was not reduced significantly compared to the activity of the wild-type strain. AAT1 activity was reduced by 50% when tyrosine was used as the amino acid donor, whereas there was a 30% reduction when tryptophan was used, compared to the activity of the wild-type strain. Expression of the fusion protein was up-regulated in both logarithmic and stationary phases by several compounds, including IAA, tryptophan, tyrosine, and phenyl acetic acid. We observed the expression of hisC1 in bacteria associated with wheat roots. Root exudates of wheat and maize were able to stimulate hisC1 expression.

Conclusions

The expression data indicate that hisC1 is under a positive feedback control in the presence of root exudates and on plants, suggesting that AAT1 activity plays a role in Azospirillum–plant interactions.     

Role of FAP48 in HIV-associated lipodystrophy

The highly active antiretroviral therapy (HAART) can cause a metabolic syndrome consisting of lipodystropy/lipoatrophy, dyslipidemia, and type 2 diabetes mellitus with an increased cardiovascular risk. The pathogenetic bases of HAART‐associated lipodystrophy are poorly known. A genetic screen was used to evaluate proteins that are modulated in HIV‐1‐infected patients with or without lipodystrophy syndrome, that are routinely treated with HAART regimens. The most significant modulation was represented by FAP48 expression. Stable over‐expression of FAP48 was able to alter, in vitro, adipogenesis, acting both on calcineurin and glucocorticoid pathways. Finally, we demonstrated that FAP48 over‐expression was able to influence the capacity of some HIV drugs, Saquinavir and Efavirenz, but not Stavudine, Amprenavir, and Indinavir to inhibit adipocyte formation. In conclusion, this molecule could be a potential target for novel therapeutic approaches to the HAART related lipodystrophy in HIV patients. J. Cell. Biochem. 113: 3446–3454, 2012. © 2012 Wiley Periodicals, Inc.     

Discovery and structure–activity relationship of a novel spirocarbamate series of NPY Y5 antagonists

A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u, which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration and strong preclinical profile for development. Both compounds significantly inhibited the food intake induced by a Y5 selective agonist with minimal effective doses of 3 mg/kg po.     

Proliferative characteristics and chromosomal stability of bovine donor cells for nuclear transfer

Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. Abnormal phosphorylation patterns of the histones during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine proliferative characteristics, chromosomal stability, and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation. Proliferative characteristics, percentage of aneuploid cells, and relative levels of phosphorylated histone H3 (ser10) were determined at different population doublings (PD) by cell counting, karyotyping, and flow cytometry, respectively. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. Some cell lines had up to 50% of aneuploid cells during early passages. Multinucleated cells and abnormal spindle configurations were observed after prolonged time in culture (60 and 41%, respectively). An increase in the relative level of phosphorylated histone occurred after extended time in culture (55.7 during early passages vs. 102.6 at late passages). These data demonstrate the importance of determining chromosome content and the selection of healthy cell lines to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of nuclear transfer (NT).     

Dihydrotestosterone potentiates insulin to up-regulate prokineticin-1 in decidualizing human endometrial stromal cells

Prokineticin 1 (PROK1) is a key regulator of embryo implantation and placentation, and its dysregulation is associated with pregnancy complications, such as pre-eclampsia and foetal growth restriction. We have previously shown that insulin strongly enhances the expression of PROK1 in human decidualizing stromal cells. Here, we demonstrate that dihydrotestosterone (DHT), but not testosterone, potentiates insulin to up-regulate PROK1 in these cells. However, the androgens alone do not influence the expression of PROK1. Our findings suggest that insulin and androgens both are involved in the regulation of PROK1 that could have implications for normal and pathological pregnancies.     

Phospholipase C gamma 2 is essential for specific functions of Fc epsilon R and Fc gamma R

Phospholipase Cgamma2 (PLCgamma2) plays a critical role in the functions of the B cell receptor in B cells and of the FcRgamma chain-containing collagen receptor in platelets. Here we report that PLCgamma2 is also expressed in mast cells and monocytes/macrophages and is activated by cross-linking of Fc(epsilon)R and Fc(gamma)R. Although PLCgamma2-deficient mice have normal development and numbers of mast cells and monocytes/macrophages, we demonstrate that PLCgamma2 is essential for specific functions of Fc(epsilon)R and Fc(gamma)R. While PLCgamma2-deficient mast cells have normal mitogen-activated protein kinase activation and cytokine production at mRNA levels, the mutant cells have impaired Fc(epsilon)R-mediated Ca(2+) flux and inositol 1,4,5-trisphosphate production, degranulation, and cytokine secretion. As a physiological consequence of the effect of PLCgamma2 deficiency, the mutant mice are resistant to IgE-mediated cutaneous inflammatory skin reaction. Macrophages from PLCgamma2-deficient mice have no detectable Fc(gamma)R-mediated Ca(2+) flux; however, the mutant cells have normal Fc(gamma)R-mediated phagocytosis. Moreover, PLCgamma2 plays a nonredundant role in Fc(gamma)R-mediated inflammatory skin reaction.     

Biochemical and Immunological Properties of the Mouse Brain Enolases Purified by a Simple Method

Abstract: A simple and rapid purification method is presented for the two mouse cerebral isozymes of enolase (EC 4.2.1.11), E₁ and E₃. The purity of the preparations was ascertained by electrophoresis under two different conditions. The biochemical and immunological properties of E₁ and E₃ were compared. The molecular weight of the cerebral enolases was analysed by column chromatography on Sephadex G 150 and by electrophoresis in the presence of SDS. Both E₁ and E₃ are homodimers with a subunit of molecular weight of 50,000. The procedure also yields a semi-purified fraction of E₂. Conditions of in vitro formation of E₂ from pure or semi-purified fractions of E₁ and E₃ show that it is likely to be a real hybrid, rather than an aggregate and that it is probably not an artefact formed during the purification. The K_m values (K_m= 3–4·10^?5 M) for the substrate are not significantly different amongst the three forms. However, E₁ and E₂ but not E₃ are inhibited by excess substrate. Antisera against E₁ and E₃ have been obtained from rabbit and goat, respectively. Antibodies against each protein do not show any cross-reactivity with each other. There is, however, a broad species cross-reactivity, showing conservation of each enolase form during evolution. Both anti-E₁ and anti-E₃ sera react with the E₂ enolase fraction, in agreement with its hybrid structure. Anti-E₃ serum does not react with extracts of other tested organs. Brain enolase 1 resembles liver enolase in its biochemical and immunological properties. A slight cross-reactivity of anti-E₁ serum with muscle extracts is observed. Heterogeneity of brain enolase 1 is observed by both biochemical and immunological methods; the nature of this heterogeneity is discussed.     

Immunoglobulin superantigen protein L induces IL-4 and IL-13 secretion from human Fc epsilon RI+ cells through interaction with the kappa light chains of IgE

Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1-B5) that interact with the variable domain of Ig kappa L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (r(s) = 0.82; p < 0.001) between the maximal percent IL-4 release induced by protein L and that induced by anti-IgE and between intact protein L and the B1-B4 fragment (r(s) = 0.90; p < 0.01). Removal of IgE bound to basophils markedly reduced the IL-4 release induced by anti-IgE, protein L, and B1-B4. Preincubation of basophils with protein L or anti-IgE caused complete cross-desensitization to subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda chains) blocked anti-IgE-induced IL-4 release, but not the releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with kappa L chains of the IgE isotype.