Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.     

Immune risk phenotype is associated with nosocomial lung infections in elderly in-patients

Background

Nosocomial infections are extremely common in the elderly and may be related to ageing of the immune system. The Immune Risk Phenotype (IRP), which predicts shorter survival in elderly patients, has not been evaluated as a possible risk factor for nosocomial infection. Our aim was to assess the prevalence of nosocomial infections in elderly in-patients and to investigate potential relationships between nosocomial infections and the immunophenotype, including IRP parameters.

Results

We included 252 consecutive in-patients aged 70 years or over (mean age, 85 ± 6.2 years), between 2006 and 2008. Among them, 97 experienced nosocomial infections, yielding a prevalence rate of 38.5% (95% confidence interval, 32.5-44.5). The main infection sites were the respiratory tract (21%) and urinary tract (17.1%) When we compared immunological parameters including cell counts determined by flow cytometry in the groups with and without nosocomial infections, we found that the group with nosocomial infections had significantly lower values for the CD4/CD8 ratio and naive CD8 and CD4 T-cell counts and higher counts of memory CD8 T-cells with a significant increase in CD28-negative CD8-T cells. Neither cytomegalovirus status (positive in 193/246 patients) nor presence of the IRP was associated with nosocomial infections. However, nosocomial pneumonia was significantly more common among IRP-positive patients than IRP-negative patients (17/60 versus 28/180; p = 0.036).

Conclusion

Immunological parameters that are easy to determine in everyday practice and known to be associated with immune system ageing and shorter survival in the elderly are also associated with an elevated risk of nosocomial pneumonia in the relatively short term.     

Immediate early gene X1 (IEX-1) is organized in subnuclear structures and partially co-localizes with promyelocytic leukemia protein in HeLa cells

Immediate early gene X1 (IEX-1) represents a stress response gene involved in growth control and modulation of apoptosis. Here, we report a detailed analysis of IEX-1 with respect to its intracellular localization. By means of confocal laser scanning microscopy, a green fluorescent protein-IEX-1 fusion protein transfected into HeLa cells, as well as endogenous IEX-1, could be detected in distinct subnuclear structures. This particular subnuclear localization of IEX-1 was not observed with a green fluorescent protein-IEX-1 fusion protein lacking a putative nuclear localization sequence, along with a decreased effect on apoptosis. Double immunofluorescence staining revealed a partial co-localization of endogenous promyelocytic leukemia protein (PML) and IEX-1 in these subnuclear structures. Nuclear localization of IEX-1 is also enhanced upon treatment of cells with leptomycin B, an inhibitor of the nuclear exporter CRM1. These observations indicate that IEX-1 is specifically shuttled to and from the nucleus. Overexpression experiments using PML isoforms III and IV revealed distinct intranuclear interaction of IEX-1 and PML. Coprecipitation experiments showed physical interaction between IEX-1 and PML. The close structural relation of IEX-1-containing nuclear subdomains and PML nuclear bodies suggests a function of IEX-1 related to the multiple functions of these unique subnuclear regions, particularly during stress response and growth control.     

Mouse erythrocyte carriers osmotically loaded with methotrexate

The mouse red blood cell (RBC) and red blood cell ghost (RBCG) have been studied as carriers of methotrexate (MTX). When incubated with high concentrations of MTX, RBCs take up significant quantities of it. However, when active loading techniques, such as the slow dialysis and preswell methods, are applied to those cells, up to 15 times more MTX can be entrapped. We have studied factors critical to the incorporation, leakage, and morphology of RBCGs during their loading with MTX by the slow dialysis and preswell methods. Compounds added to the buffers to maintain the ATP content of the cells and osmolarity play functional roles in this process. The fate of the material entrapped within the ghosts after in vivo administration was shown to be capture by the reticuloendothelial system. The pharmacological efficacy of MTX-loaded RBCGs in treating mice bearing hepatoma ascites tumors was demonstrated by increases in average survival time of 28.5-42.8%.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Estimating haplotype relative risks on human survival in population-based association studies

Association-based linkage disequilibrium (LD) mapping is an increasingly important tool for localizing genes that show potential influence on human aging and longevity. As haplotypes contain more LD information than single markers, a haplotype-based LD approach can have increased power in detecting associations as well as increased robustness in statistical testing. In this paper, we develop a new statistical model to estimate haplotype relative risks (HRRs) on human survival using unphased multilocus genotype data from unrelated individuals in cross-sectional studies. Based on the proportional hazard assumption, the model can estimate haplotype risk and frequency parameters, incorporate observed covariates, assess interactions between haplotypes and the covariates, and investigate the modes of gene function. By introducing population survival information available from population statistics, we are able to develop a procedure that carries out the parameter estimation using a nonparametric baseline hazard function and estimates sex-specific HRRs to infer gene-sex interaction. We also evaluate the haplotype effects on human survival while taking into account individual heterogeneity in the unobserved genetic and nongenetic factors or frailty by introducing the gamma-distributed frailty into the survival function. After model validation by computer simulation, we apply our method to an empirical data set to measure haplotype effects on human survival and to estimate haplotype frequencies at birth and over the observed ages. Results from both simulation and model application indicate that our survival analysis model is an efficient method for inferring haplotype effects on human survival in population-based association studies.     

PhySIC_IST: cleaning source trees to infer more informative supertrees

Background  

Supertree methods combine phylogenies with overlapping sets of taxa into a larger one. Topological conflicts frequently arise among source trees for methodological or biological reasons, such as long branch attraction, lateral gene transfers, gene duplication/loss or deep gene coalescence. When topological conflicts occur among source trees, liberal methods infer supertrees containing the most frequent alternative, while veto methods infer supertrees not contradicting any source tree, i.e. discard all conflicting resolutions. When the source trees host a significant number of topological conflicts or have a small taxon overlap, supertree methods of both kinds can propose poorly resolved, hence uninformative, supertrees.     

Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols

The mechanism-based inhibition of dopamine beta-hydroxylase (DBH; EC 1.14.17.1) by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding [DeWolf, W. E., Jr., & Kruse, L. I. (1985) Biochemistry 24, 3379]. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition [Ahn, N., & Klinman, J. P. (1983) Biochemistry 22, 3096] p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate "activator" addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible, is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.     

Comparison of the cytomorphological classification with the cytochemical differentiation of acute lymphatic leukemia in children]

In 100 children with acute lymphatic leukaemia the cytomorphological subclassification of the pathological cell type was made according to Mathé and the French-American-British Co-operative group (FAB). In addition, all cases of leukaemia were differentiated according to their cytochemical type. Lymphoblasts from 10 cases of leukaemia could be subclassified immunologically. From 71 children will ALL the survival rates of those cases of leukaemia subclassified cytomorphologically and the cytochemical reactions were compiled and partially compared. Microlymphoblastic leukaemia could be found to be the most frequent type of ALL at children's age. Prolymphocytic leukaemias were characterized by a favourable survival rate and the highest percentage of ALL with the PAS type. Macrolymphoblastic and microlymphoblastic cases of leukaemia revealed no essential differences of survival rate, but significant differences of cytochemical reactions.