Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.     

Glycosylation enhances functional stability of the chemotactic cytokine CCL2

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions.     

IL-12Rbeta2-deficient mice of a genetically resistant background are susceptible to Leishmania major infection and develop a parasite-specific Th2 immune response

The cytokine IL-12 plays a critical role in inducing the production of IFN-gamma from T and NK cells and in the polarization of T cells towards the Th1 phenotype. IL-12 is comprised of two subunits (IL-12p40 and IL-12p35) that together form the biologically active p70 molecule, and IL-12 functions via binding to a heterodimeric receptor (IL-12Rbeta1 and IL-12Rbeta2). Previous studies utilizing mice deficient for either the IL-12 cytokine or the IL-12-induced signaling molecule STAT4 have established a critical role for IL-12 during infection with Leishmania major. However, these studies warrant careful re-interpretation in light of the recent discovery of the IL-12-related cytokine, IL-23, which utilizes the IL-12p40 chain in combination with an IL-12p35-related molecule, called p19, and a receptor comprised of the IL-12Rbeta1 chain plus a unique chain referred to as IL-23R. We analyzed the course of L. major infection in mice deficient for the IL-12-specific IL-12Rbeta2 subunit in order to assess the role of IL-12 signaling without disruption of the IL-23 pathway. After infection with L. major, IL-12Rbeta2KO mice of a resistant background (C57Bl/6) developed large cutaneous lesions similar to those developed by susceptible BALB/c mice. Draining lymph node cells from L. major-infected IL-12Rbeta2KO mice released the Th2 cytokines IL-4 and IL-5 after in vitro stimulation with Leishmania lysate but were completely devoid of IFN-gamma, consistent with a default towards a strong parasite-specific Th2 response. L. major-infected IL-12Rbeta2KO mice were also devoid of parasite-specific IgG2a antibodies, and interestingly, their footpad lesions ulcerated earlier than those of susceptible BALB/c mice.     

A role for the exosome in the in vivo degradation of unstable mRNAs

In mammals, the mRNAs encoding many proteins involved in inflammation bear destabilizing AU-rich elements (AREs) in the 3'-untranslated region. The exosome, a complex of 3' --> 5' exonucleases, is rate limiting in the destruction of such mRNAs in a mammalian in vitro system, but a role in vivo has not been demonstrated. The phenomenon of ARE-mediated degradation also occurs in the protist parasite Trypanosoma brucei. Messenger RNAs with 3'-untranslated region U-rich elements, which strongly resemble AREs, are extremely unstable in the trypanosome form that parasitizes mammals. The first step in degradation of these mRNAs in vivo is rapid destruction of the 3'-untranslated region; subsequently the mRNA is destroyed by exonucleases acting in both 5' --> 3' and 3' --> 5' directions. We here investigated the roles of three subunits of the trypanosome exosome complex, RRP45, RRP4, and CSL4, in this process, depleting the individual subunits in vivo by inducible RNA interference. RRP45 depletion, which probably disrupts exosome integrity, caused a delay in the onset of degradation of the very unstable RNAs, but did not affect degradation of more stable species. Depletion of RRP4 or CSL4 does not affect the stability of the residual exosome and did not change mRNA degradation kinetics. We conclude that the exosome is required for the initiation of rapid degradation of unstable mRNAs in trypanosomes.     

Susceptibility of non-linear systems as an approach to metabolic responses

MOTIVATION: Recent developments of several analytical techniques and fast sampling procedures are making it possible to study non-linear dynamics on an automated basis. Approaches that suggest experimental setups and fully exploit the data are needed. To prevent unreliable fits of parameters to experimental data, model-independent methods would be advantageous. One such method consists in representing the response of a system by means of a transformation or functional, of an excitation, i.e. a flux of a metabolite. RESULTS: A functional of unknown form can be expanded in series if its functional derivatives are known. An algorithm for calculating such generalized derivatives from impulse-perturbation experiments was developed. The only assumption was that the implicit kinetics is time-independent, i.e. the system is time-invariant. The method is illustrated on a part of fructose catabolism. A reaction network that involves 14 metabolites and 11 enzymes and includes several branches and feedback loops is considered. It is shown that the method provides good approximations to the responses of this complex system. AVAILABILITY: FUNDER is available from http://bbm1.ucm.es/torralba/funder/down/ SUPPLEMENTARY INFORMATION: http://bbm1.ucm.es/torralba/funder/     

Identification and characterization of the<Emphasis Type="Italic"> carAB</Emphasis> genes responsible for encoding carbamoylphosphate synthetase in<Emphasis Type="Italic"> Halomonas eurihalina</Emphasis>

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.     

Pseudocysts in trichomonads--new insights

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with anti-tubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.     

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.     

GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.     

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.