TGF-β induces the expression of SAP30L, a novel nuclear protein

Background  

We have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-β (TGF-β), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-β-induced differentiation.     

Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris.

Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional (13)C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h(-1), 0.16 h(-1)) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h(-1) on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h(-1) (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h(-1). Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h(-1). This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.     

Genetic polymorphism of the human ICOS gene

Inducible costimulator (ICOS) is a novel receptor belonging to the same family as CD28 and CTLA4, which regulate T-lymphocyte activation in the immune response. The genes for these molecules are located adjacent to each other on Chromosome 2q33. Many autoimmune diseases have been found to be genetically linked to or associated with genetic markers near the CTLA4 gene. However, as all three genes are closely linked and have related functions, it is possible that the findings could be explained by variation in CD28 or ICOS. Few data on genetic variation in the ICOS gene are available. We sequenced the ICOS gene in 13 healthy unrelated individuals and found eight single nucleotide polymorphisms. One was located in the first intron, and the others in the untranslated region of the last exon. The allele frequencies and linkage disequilibrium were determined from a population sample of 63 Finnish individuals. The results show that the ICOS gene is polymorphic, but no variation in the coding sequence was detected, implying that the genetic linkage of this gene region to autoimmune diseases may not result from structural variation in the ICOS molecule. These polymorphisms, however, should be useful in genetic studies of this candidate gene.     

Extracellular transglutaminase 2 has a role in cell adhesion, whereas intracellular transglutaminase 2 is involved in regulation of endothelial cell proliferation and apoptosis

Objective: Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell–matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi. Materials and methods: The effect of small interfering RNA (siRNA)‐TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V/PI dual staining and protein expression level was assayed by western blotting. Results: We found that siRNA‐TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G₁ phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA‐TG2‐induced apoptosis. Conclusion: We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro.     

Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation

Extremophiles - Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses...     

Structural flexibility allows the functional diversity of potyvirus genome-linked protein VPg

Several viral genome-linked proteins (VPgs) of plant viruses are intrinsically disordered and undergo folding transitions in the presence of partners. This property has been postulated to be one of the factors that enable the functional diversity of the protein. We created a homology model of Potato virus A VPg and positioned the known functions and structural properties of potyviral VPgs on the novel structural model. The model suggests an elongated structure with a hydrophobic core composed of antiparallel β-sheets surrounded by helices and a positively charged contact surface where most of the known activities are localized. The model most probably represents the fold induced immediately after binding of VPg to a negatively charged lipid surface or to SDS. When the charge of the positive surface was lowered by lysine mutations, the efficiencies of in vitro NTP binding, uridylylation reaction, and unspecific RNA binding were reduced and in vivo the infectivity was debilitated. The most likely uridylylation site, Tyr63, locates to the positively charged surface. Surprisingly, a Tyr63Ala mutation did not prevent replication completely but blocked spreading of the virus. Based on the localization of Tyr119 in the model, it was hypothesized to serve as an alternative uridylylation site. Evidence to support the role of Tyr119 in replication was obtained which gives a positive example of the prediction power of the model. Taken together, our experimental data support the features presented in the model and the idea that the functional diversity is attributable to structural flexibility.     

Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates.