LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of <Emphasis Type="Italic">Leishmania mexicana</Emphasis>

Background  

Leishmania parasites undergo profound morphological and biochemical changes while passing through their life cycle. Protein kinases have been shown to be involved in the differentiation from the extracellular flagellated promastigotes to the intracellular "non-flagellated" amastigotes and vice versa. Moreover, these enzymes are likely involved in the regulation of the proliferation of the different life stages.     

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.     

The identification of 1,3-oxazolidine-2-thiones and 1,3-thiazolidine-2-thiones from the reaction of glucose with benzyl isothiocyanate

The structure of interaction products resulting from the reaction of unmodified glucose with benzyl isothiocyanate is reported. Prior to their identification, the main products of this reaction were isolated using solid-phase extraction (SPE) as well as preparative HPLC. They were then identified by NMR and MS as 3-benzyl-4-hydroxy-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-oxazolidine-2-thione, 3-benzyl-4-hydroxy-4-hydroxymethyl-5-(D-erythro-1,2,3-trihydroxypropyl)-1,3-oxazolidine-2-thione, N-benzyl-(D-gluco-4,5-dihydroxy-6-hydroxymethyl-tetrahydropyrano)[2,3-b]oxazolidine-2-thione and 3-benzyl-4-(N-benzyl amino)-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-thiazolidine-2-thione. The identity of the last compound was secured by X-ray crystal structure data.     

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.     

The Interplay of Proton,Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii

To obtain a detailed picture of sulfur deprivation-induced H₂ production in microalgae, metabolome analyses were performed during key time points of the anaerobic H₂ production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H₂ production process. This work reports on the differential analysis of metabolic profiles of the high H₂-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H₂ production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H₂ production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.     

Genetic diversity and description of transmission routes for Campylobacter on broiler farms by amplified-fragment length polymorphism

AIMS: To investigate the genetic diversity of Campylobacter in broilers and in the environment of broiler farms, to compare the genetic profiles and describe critical factors for transmission to broilers. METHODS AND RESULTS: Flocks at three of four investigated farms became colonized with Campylobacter. The total proportion of Campylobacter-positive samples at different farms varied from 20% to 42%. The farm with the poorest biosecurity routines had broilers that became infected earliest, the highest proportion of positive samples and the highest genetic diversity among the broiler Campylobacter isolates. Campylobacter isolates within common amplified-fragment length polymorphism (AFLP) clusters (95-100%) were found to be present in outdoor environment and in broilers at adjacent farms before they were found in the broilers. A large presence of Campylobacter in the farm environment was demonstrated after the broilers were infected. A high genetic diversity was found among Campylobacter present in the outdoor environment, where certain Campylobacter clusters were found for periods of up to 6 weeks. CONCLUSION: Confirmation by AFLP indicates adjacent poultry farms and outdoor environment as major sources of Campylobacter infection of broilers, this being the novel achievements. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide more exact knowledge on transmission of Campylobacter at farm level, helpful for developing optimal preventive strategies.     

Prevalence, persistence, and molecular characterization of glycopeptide-resistant enterococci in Norwegian poultry and poultry farmers 3 to 8 years after the ban on avoparcin

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant Enterococcus faecium (GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn1546-plasmid junction fragment in 93.9% of E. faecium isolates. A putative postsegregation killing (PSK) system linked to Tn1546 was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (n = 10) and -resistant (n = 10) E. faecium isolates from humans (n = 10) and poultry (n = 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated vanA-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.     

Genetic association analysis of human longevity in cohort studies of elderly subjects: an example of the PON1 gene in the Danish 1905 birth cohort

Although the case-control or the cross-sectional design has been popular in genetic association studies of human longevity, such a design is prone to false positive results due to sampling bias and a potential secular trend in gene-environment interactions. To avoid these problems, the cohort or follow-up study design has been recommended. With the observed individual survival information, the Cox regression model has been used for single-locus data analysis. In this article, we present a novel survival analysis model that combines population survival with individual genotype and phenotype information in assessing the genetic association with human longevity in cohort studies. By monitoring the changes in the observed genotype frequencies over the follow-up period in a birth cohort, we are able to assess the effects of the genotypes and/or haplotypes on individual survival. With the estimated parameters, genotype- and/or haplotype-specific survival and hazard functions can be calculated without any parametric assumption on the survival distribution. In addition, our model estimates haplotype frequencies in a birth cohort over the follow-up time, which is not observable in the multilocus genotype data. A computer simulation study was conducted to specifically assess the performance and power of our haplotype-based approach for given risk and frequency parameters under different sample sizes. Application of our method to paraoxonase 1 genotype data detected a haplotype that significantly reduces carriers' hazard of death and thus reveals and stresses the important role of genetic variation in maintaining human survival at advanced ages.     

Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy

In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.