Direct analysis of the extracellular proteome from two strains of Helicobacter pylori

Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N-acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins.     

Maßgeschneiderte Mikroorganismen

Tailor‐made microorganisms Microbial diversity provides unlimited resources for the development of novel industrial processes and products. Since the beginning of the 20th century microorganisms have been successfully applied for the large scale production of bio‐based products. In recent years, modern methods of strain development and Synthetic Biology have enabled biotech engineers to design even more sophisticated and tailor‐made microorganisms. These microbes serve industrial processes for the production of bulk chemicals, enzymes, polymers, biofuels as well as plant‐derived ingredients such as Artemisinin in an ecologically and economically sustainable and attractive fashion. In the future, production of advanced biofuels, microbial fuel cells, CO₂ as feedstock and microbial cellulose are research topics as well as challenges of global importance. Continuous efforts in microbiology and biotechnology research will be pivotal for white biotechnology to gain more momentum in transforming the chemical industry towards a knowledge based bio‐economy.     

Enumeration of the simian virus 40 early region elements necessary for human cell transformation

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.     

Proposal for Naming Host Cell-Derived Inserts in Retrovirus Genomes

We propose a system for naming inserted sequences in transforming retroviruses (i.e., onc genes), based on using trivial names derived from a prototype strain of virus.     

The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3

The common cytokine receptor gamma chain (gamma c), an essential component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. Recently, a novel lymphokine (IL-21) and its receptor (IL-21R alpha) were described which profoundly affect the growth and activation state of B, T, and NK cells in concert with other lymphokines or stimuli [Parrish-Novak, J., et al. (2000) Nature 408, 57-63]. In this report, we show that gamma c is also a required signaling component of the IL-21 receptor (IL-21R) using the gamma c-deficient X-linked severe combined immunodeficiency (XSCID) lymphoblastoid cell line JT, and JT cells reconstituted with gamma c (JT/gamma c). Moreover, we demonstrate a functional requirement for both gamma c and the gamma c-associated Janus family tyrosine kinase 3 (JAK3) in IL-21-induced proliferation of pro-B-lymphoid cells engineered to express human IL-21R alpha (BaF3/IL-21R alpha). Retroviral-mediated transduction of wild-type gamma c into XSCID JT cells restored function to the IL-21R, as shown by IL-21-induced tyrosine phosphorylation of JAK1 and JAK3, and downstream activation of STAT5, in JT/gamma c cells as well as BaF3/IL-21R alpha and primary splenic B cells. In contrast, IL-21 failed to activate the JAK-STAT pathway in nonreconstituted JT cells. Monoclonal antibodies specific for the gamma c chain effectively inhibited IL-21-induced growth of BaF3/IL-21R alpha cells, supporting a functional role for this molecule in the IL-21R complex. In addition, the specific JAK3 tyrosine kinase inhibitor WHI-P131 significantly reduced IL-21-induced proliferation of BaF3/IL-21R alpha cells. Taken together, these results definitively demonstrate that IL-21-mediated signaling requires the gamma c chain, and indicate that JAK3 is an essential transducer of gamma c-dependent survival and/or mitogenic signals induced by this cytokine.     

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

The effect of a propionic acid bacterial inoculant applied at ensiling on the aerobic stability of wheat and sorghum silages

The effect of a new strain ofPropionibacterium shermanii (PAB), applied at ensiling, on the aerobic stability of wheat and sorghum silages was studied in several experiments under laboratory conditions. In the one experiment with wheat and in those with sorghum a lactic acid bacteria (LAB) inoculant (Lactobacillus plantarum andPediococcus cerevisiae) was also included. After treatment, the chopped forages were ensiled in 1.5-L anaerobic jars which were sampled in triplicate on predetermined dates to follow fermentation dynamics. At the end of the experiments, the silages were subjected to an aerobic stability test. The PAB inoculant improved the aerobic stability only in one experiment with wheat, in which the decrease in pH was very slow; the final pH remained relatively high (4.5). The PAB-treated silages contained 19.5±2.0 g of propionic acid per kg of dry matter. In the experiments with sorghum, the control and PAB-inoculated silages were stable, whereas LAB-inoculated silages deteriorated. The results suggest that PAB can survive in and improve the aerobic stability of only slow-fermenting silages which are prone to aerobic deterioration.     

Clinical and Biological Relevance of Genomic Heterogeneity in Chronic Lymphocytic Leukemia

Background

Chronic lymphocytic leukemia (CLL) is typically regarded as an indolent B-cell malignancy. However, there is wide variability with regards to need for therapy, time to progressive disease, and treatment response. This clinical variability is due, in part, to biological heterogeneity between individual patients’ leukemias. While much has been learned about this biological variation using genomic approaches, it is unclear whether such efforts have sufficiently evaluated biological and clinical heterogeneity in CLL.

Methods

To study the extent of genomic variability in CLL and the biological and clinical attributes of genomic classification in CLL, we evaluated 893 unique CLL samples from fifteen publicly available gene expression profiling datasets. We used unsupervised approaches to divide the data into subgroups, evaluated the biological pathways and genetic aberrations that were associated with the subgroups, and compared prognostic and clinical outcome data between the subgroups.

Results

Using an unsupervised approach, we determined that approximately 600 CLL samples are needed to define the spectrum of diversity in CLL genomic expression. We identified seven genomically-defined CLL subgroups that have distinct biological properties, are associated with specific chromosomal deletions and amplifications, and have marked differences in molecular prognostic markers and clinical outcomes.

Conclusions

Our results indicate that investigations focusing on small numbers of patient samples likely provide a biased outlook on CLL biology. These findings may have important implications in identifying patients who should be treated with specific targeted therapies, which could have efficacy against CLL cells that rely on specific biological pathways.     

Cellular regulation of iron assimilation

Cells of plants, most microorganisms, and animals require well-defined amounts of iron for survival, replication, and differentiation. The metal is an important component of such processes as synthesis of DNA, RNA, and chlorophyll; electron transport; oxygen metabolism; and nitrogen fixation. Because of the insolubility of iron in aerobic environments at neutral and alkaline pH values, cells have had to devise specific strategies to assimilate the metal. These include (1) development of systems for reducing ferric ions to the more soluble ferrous ions at the cell surface, (2) employment of small carrier molecules (termed siderophores) that have high affinity for ferric ions and receptor proteins for the ferrated molecules, and (3) use of transferrin and other proteins that can transport ferric ions. Excessive amounts of iron are toxic, however, and intracellular storage capacity is limited and efflux mechanisms generally are lacking. Thus, cells have had to develop methods of preventing over-accumulation of the metal. These include use of (1) oxygen to convert ferrous to ferric ions, (2) small molecules that can bind ferrous ions, termed siderophraxes, and (3) proteins that, when combined with ferrous ions, repress the expression of iron transport genes. Often, one organism can prevent growth of neighbors by restricting their access to iron. In other cases, cells assist each other by sharing iron acquisition systems or by restricting influx of excess iron. Homeostatic control of other essential trace metals also is required for optimal cell function. Nevertheless, since iron thus far has received most attention, it serves as the model of mineral metabolism. Moreover, many of the observations made on control of iron metabolism suggest possible applications in prevention and management of plant and animal infections as well as of neoplastic diseases, arthropathy, and cardiomyopathy. This review will focus on (1) problems at the cellular level of iron acquisition, storage, and exclusion; and (2) the strategies devised by cells of plants, microorganisms, and animals to solve these problems.