Direct random insertion mutagenesis of Helicobacter pylori

Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or instability of the cloned DNA, or toxicity of the encoded products. We therefore created two mutant libraries in the human pathogen Helicobacter pylori using a simple, direct mutagenesis technique, which does not require E. coli as intermediate. H. pylori total DNA was digested, circularized and digested again with a frequently cutting restriction enzyme, and the resulting fragments were ligated to a kanamycin antibiotic resistance cassette. Subsequently, the ligation mixture was transformed into the parental H. pylori strain 1061. Insertion of the kanamycin cassette by double homologous recombination into the genome of H. pylori 1061 resulted in approximately 2500 kanamycin resistant colonies. Heterogeneity of kanamycin cassette insertion was confirmed by Southern blotting. The isolation of two independent H. pylori mutants defective in production of urease from this library underlines the potential of this mutagenesis strategy.     

Thyroid status,but not insulin status,affects expression of avian uncoupling protein mRNA in chicken

The aim of this study was to investigate the hormonal regulation of the avian homolog of mammalian uncoupling protein (avUCP) by studying the impact of thyroid hormones and insulin on avUCP mRNA expression in chickens (Gallus gallus). For 3 wk, chicks received either a standard diet (control group), or a standard diet supplemented with triiodothyronine (T(3); T3 group) or with the thyroid gland inhibitor methimazole (MMI group). A fourth group received injections of the deiodinase inhibitor iopanoic acid (IOP group). During the 4th wk of age, all animals received two daily injections of either human insulin or saline solution. The results indicate a twofold overexpression of avUCP mRNA in gastrocnemius muscle of T3 birds and a clear downregulation (-74%) in MMI chickens compared with control chickens. Insulin injections had no significant effect on avUCP mRNA expression in chickens. This study describes for the first time induction of avUCP mRNA expression by the thermogenic hormone T(3) in chickens and supports a possible involvement of avUCP in avian thermogenesis.     

CO2 efflux from a Mediterranean semi-arid forest soil. I. Seasonality and effects of stoniness

We studied the seasonality of total soil CO₂efflux and labeled C-CO₂ released from ¹⁴Clabeled straw incubated in the H horizon of asemi-arid Mediterranean forest soil. Fieldmeasurements were carried out over 520 days in aseries of reconstructed soil profiles with and withouta gravel layer below the H horizon. We monitored soilclimate and related this to soil CO₂ efflux.Seasonal variations in soil CO₂ efflux in asemiarid Mediterranean forest were mainly related tochanges in soil temperature. In spite of drought, highrespiration rates were observed in mid summer. Highsoil CO₂ efflux in hot and dry episodes wasattributed to increases in soil biological activity.The minimum soil CO₂ efflux occurred in latesummer also under dry conditions, probably related toa decrease in soil biological activity in deephorizons. Biological activity in organic layers waslimited by water potential () in summer and bytemperature in winter. Rewetting a dry soil resultedin large increases in soil CO₂ efflux only at hightemperatures. These large increases represented asignificant contribution to the decomposition oforganic matter in the uppermost horizons. Soilbiological activity in the uppermost horizons was moresensitive to changes in soil and hence tosummer rainstorms than the bulk soil microbialactivity. The presence of a layer of gravel improvedboth moisture and temperature conditions for thedecomposition of organic matter. As a result, soilCO₂ efflux increased in soils containing rockfragments. These effects were especially large for theorganic layers.     

Immunolocalization of the Vascular Endothelial Growth Factor Receptor-2 in the Subepicardial Mesenchyme of Hamster Embryos: IDentification of the Coronary Vessel Precursors

The earliest evIDence of the development of the cardiac vessels in mammals is the emergence of subepicardial blood islands, which are thought to originate from mesenchymal progenitors. In order to IDentify these progenitor cells, we have studied the immunohistochemical localization in the heart of Syrian hamster embryos of the type 2 vascular endothelial growth factor receptor, the earliest molecule known to be expressed in the vasculogenic cell lineage. Only a few immunoreactive subepicardial mesenchymal cells were present by 10 days post coitum. By 11 days post coitum, the subepicardial mesenchymal cells became abundant at the dorsal part of the ventricle, the atrioventricular and the conoventricular grooves. About 20% of cells were labelled with the antibody. Immunoreactive cells were isolated or formed pairs, short cords, rounded clusters or ring-like structures at the subepicardium or, occasionally, within the ventricular myocardium. Other labelled cells were simultaneously cytokeratin immunoreactive. By 12 days post coitum, most immunoreactive mesenchymal cells have been replaced by a capillary network. We propose that an active process of vascular differentiation occurs between 10 and 12 days post coitum in the subepicardium of this species, and it might be a suitable model for the study of vasculogenetic mechanisms.     

Growth of MDA-MB-231 cell line: different effects of TGF-beta(1), EGF and estradiol depending on the length of exposure.

The human cell line MDA-MB-231 is a prototype for the study of hormone-independent breast cancer. Modification of cell growth behaviour has been observed after treating these cells with growth factors. EGF is a typical stimulatory growth factor for many cell types, whereas transforming growth factor beta(1)(TGF-beta(1)) acts with inhibitory character. Here we observed cell growth inhibition after EGF as well as after TGF-beta(1)treatments. Nevertheless, in the 42-h experiments, EGF-treated cultures grew before (18 hours) respect to the TGF-beta(1)and E(2)-treated cultures (24 h), and in the 11-day experiments, EGF-treated cultures started growing (7 days) after TGF-beta(1)-treated cultures (5 days). Estradiol inhibited the proliferation of these cells only after several days of treatment.     

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

BackgroundLeishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection.Conclusions/SignificanceDespite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the LbrPGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.     

Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.     

Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A‐rule. In this study, we present X‐ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non‐instructive lesions by DNA polymerases at least from this sequence family.     

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.