Ampk regulates IgD expression but not energy stress with B cell activation.

Ampk is an energy gatekeeper that responds to decreases in ATP by inhibiting energy-consuming anabolic processes and promoting energy-generating catabolic processes. Recently, we showed that Lkb1, an understudied kinase in B lymphocytes and a major upstream kinase for Ampk, had critical and unexpected roles in activating naïve B cells and in germinal center formation. Therefore, we examined whether Lkb1 activities during B cell activation depend on Ampk and report surprising Ampk activation with in vitro B cell stimulation in the absence of energy stress, coupled to rapid biomass accumulation. Despite Ampk activation and a controlling role for Lkb1 in B cell activation, Ampk knockout did not significantly affect B cell activation, differentiation, nutrient dynamics, gene expression, or humoral immune responses. Instead, Ampk loss specifically repressed the transcriptional expression of IgD and its regulator, Zfp318. Results also reveal that early activation of Ampk by phenformin treatment impairs germinal center formation but does not significantly alter antibody responses. Combined, the data show an unexpectedly specific role for Ampk in the regulation of IgD expression during B cell activation

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mtl) were used to develop a digoxigenin-labeled probe by PCR, Using this probe, ale isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively, The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa, The amino acid sequence shows high similarity with the sequence of IICBMtl of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coil by placing it behind the strong tac promoter, The rate of thermal inactivation at 60 degrees C of B. stearothermophilus IICBMtl expressed in E. coli was two times lower than that of E. coli IICBMtl, IICBMtl in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.</p

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIAMtl. Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EIIMtl were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genesmtlA,mtlR,mtlF, andmtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICBMtl of S. carnosus and the IICB part of the IICBAMtls of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60&amp;C of B. stearothermophilus IICBMtl expressed in E. coli was two times lower than that of E. coli IICBMtl. IICBMtl in B. stearothermophilus is maximally active at 85&amp;C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95 % purity after six histidines were fused to the C-terminal part of the transporter. Many bacteria transport D-mannitol and other carbohy-drates via a phosphoenolpyruvate (PEP)-dependent phospho

Narrative-based computational modelling of the Gp130/JAK/STAT signalling pathway.

BACKGROUND: Appropriately formulated quantitative computational models can support researchers in understanding the dynamic behaviour of biological pathways and support hypothesis formulation and selection by "in silico" experimentation. An obstacle to widespread adoption of this approach is the requirement to formulate a biological pathway as machine executable computer code. We have recently proposed a novel, biologically intuitive, narrative-style modelling language for biologists to formulate the pathway which is then automatically translated into an executable format and is, thus, usable for analysis via existing simulation techniques. RESULTS: Here we use a high-level narrative language in designing a computational model of the gp130/JAK/STAT signalling pathway and show that the model reproduces the dynamic behaviour of the pathway derived by biological observation. We then "experiment" on the model by simulation and sensitivity analysis to define those parameters which dominate the dynamic behaviour of the pathway. The model predicts that nuclear compartmentalisation and phosphorylation status of STAT are key determinants of the pathway and that alternative mechanisms of signal attenuation exert their influence on different timescales. CONCLUSION: The described narrative model of the gp130/JAK/STAT pathway represents an interesting case study showing how, by using this approach, researchers can model biological systems without explicitly dealing with formal notations and mathematical expressions (typically used for biochemical modelling), nevertheless being able to obtain simulation and analysis results. We present the model and the sensitivity analysis results we have obtained, that allow us to identify the parameters which are most sensitive to perturbations. The results, which are shown to be in agreement with existing mathematical models of the gp130/JAK/STAT pathway, serve us as a form of validation of the model and of the approach itself

Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures.

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan-cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis-defined CNA signatures are predictive of glycolytic phenotypes, including 18F-fluorodeoxy-glucose (FDG) avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer-linked metabolic enzymes. A pan-cancer and cross-species comparison of CNAs highlighted 26 consistently altered DNA regions, containing 11 enzymes in the glycolysis pathway in addition to known cancer-driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the subsequent copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution

The Same IκBα Mutation in Two Related Individuals Leads to Completely Different Clinical Syndromes

Both innate and adaptive immune responses are dependent on activation of nuclear factor κB (NF-κB), induced upon binding of pathogen-associated molecular patterns to Toll-like receptors (TLRs). In murine models, defects in NF-κB pathway are often lethal and viable knockout mice have severe immune defects. Similarly, defects in the human NF-κB pathway described to date lead to severe clinical disease. Here, we describe a patient with a hyper immunoglobulin M–like immunodeficiency syndrome and ectodermal dysplasia. Monocytes did not produce interleukin 12p40 upon stimulation with various TLR stimuli and nuclear translocation of NF-κB was impaired. T cell receptor–mediated proliferation was also impaired. A heterozygous mutation was found at serine 32 in IκBα. Interestingly, his father has the same mutation but displays complex mosaicism. He does not display features of ectodermal dysplasia and did not suffer from serious infections with the exception of a relapsing Salmonella typhimurium infection. His monocyte function was impaired, whereas T cell function was relatively normal. Consistent with this, his T cells almost exclusively displayed the wild-type allele, whereas both alleles were present in his monocytes. We propose that the T and B cell compartment of the mosaic father arose as a result of selection of wild-type cells and that this underlies the widely different clinical phenotype