Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase

The matriptase-prostasin proteolytic cascade is essential for epidermal tight junction formation and terminal epidermal differentiation. This proteolytic pathway may also be operative in a variety of other epithelia, as both matriptase and prostasin are involved in tight junction formation in epithelial monolayers. However, in polarized epithelial cells matriptase is mainly located on the basolateral plasma membrane whereas prostasin is mainly located on the apical plasma membrane. To determine how matriptase and prostasin interact, we mapped the subcellular itinerary of matriptase and prostasin in polarized colonic epithelial cells. We show that zymogen matriptase is activated on the basolateral plasma membrane where it is able to cleave relevant substrates. After activation, matriptase forms a complex with the cognate matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1 and is efficiently endocytosed. The majority of prostasin is located on the apical plasma membrane albeit a minor fraction of prostasin is present on the basolateral plasma membrane. Basolateral prostasin is endocytosed and transcytosed to the apical plasma membrane where a long retention time causes an accumulation of prostasin. Furthermore, we show that prostasin on the basolateral membrane is activated before it is transcytosed. This study shows that matriptase and prostasin co-localize for a brief period of time at the basolateral plasma membrane after which prostasin is transported to the apical membrane as an active protease. This study suggests a possible explanation for how matriptase or other basolateral serine proteases activate prostasin on its way to its apical destination.     

Case-control and case-only designs with genotype and family history data: estimating relative risk, residual familial aggregation, and cumulative risk

In case-control studies of inherited diseases, participating subjects (probands) are often interviewed to collect detailed data about disease history and age-at-onset information in their family members. Genotype data are typically collected from the probands, but not from their relatives. In this article, we introduce an approach that combines case-control analysis of data on the probands with kin-cohort analysis of disease history data on relatives. Assuming a marginally specified multivariate survival model for joint risk of disease among family members, we describe methods for estimating relative risk, cumulative risk, and residual familial aggregation. We also describe a variation of the methodology that can be used for kin-cohort analysis of the family history data from a sample of genotyped cases only. We perform simulation studies to assess performance of the proposed methodologies with correct and mis-specified models for familial aggregation. We illustrate the proposed methodologies by estimating the risk of breast cancer from BRCA1/2 mutations using data from the Washington Ashkenazi Study.     

Littoral benthic macroinvertebrates of mountain lakes in the Tatra Mountains (Slovakia,Poland)

Littoral benthic macroinvertebrates of 45 mountain lakes in the Tatra Mountains were sampled using a semi-quantitative method in September 2000. A total of 32,852 specimens were identified to 93 taxa belonging to 14 higher taxonomic groups. Multivariate statistics (CCA, RDA) and nine biotic metrics (AQEM/STAR) were used to explain relationships between macroinvertebrate assemblages and environmental variables. Up to 57% of the ecological position of littoral macroinvertebrate assemblages were explained by variance of environmental variables divided into chemical, trophic, physical, catchment and location. Five types of Tatra lakes were recognized using CCA: A — strongly acidified lakes (small catchment, low pH, high concentration of TP, DOC, highest amount of POM in littoral); B — alpine acidified lakes (low amount of POM, low values of biotic metrics); C — alpine non-acidified lakes (high value of diversity index, predominance of Diptera); D — subalpine acidified lakes (high values of biotic metrics: number of families, proportion of crenal and rhithral taxa/total taxa); E — subalpine non-acidified lakes (high values of biotic metrics: number of families, number of genera, BMWP score, number of taxa and abundance of EPT taxa). RDA was used to design five levels of macroinvertebrate taxa acidification tolerance. The Tatra Acidification Index (TAI) was established to assess the acidification status of the lakes in the Tatra Mts.     

Negative effects of elevated testosterone on female fecundity in zebra finches

Although factors influencing androgen deposition in the avian egg and its effects on nestling fitness are recently receiving considerable attention, little is known about the potential costs of high testosterone levels in the females. Our study aimed at determining the effect of injections of testosterone (T) in female zebra finches (Taeniopygia guttata), on clutch size, egg mass, yolk mass, and yolk androgen content. Females were given a single bolus injection of T in a range of doses after laying the first egg. Results show that administration of T negatively affected clutch size; the strength of this effect increased with increasing doses of T. Females injected with the highest testosterone dose showed suppressed oviposition of the third and the fourth eggs. Interestingly, testosterone administration made females produce eggs with relatively large yolks, suggesting that T may mediate the trade-off between number and size of eggs. Testosterone injection resulted in elevated levels of androgen in the eggs, in contrast to control clutches, which showed a decreasing pattern of androgen concentration along the laying sequence. We conclude that high androgen investment in eggs may be limited by physiological requirements of the ovulatory process.     

Genomic inference accurately predicts the timing and severity of a recent bottleneck in a nonmodel insect population

The analysis of molecular data from natural populations has allowed researchers to answer diverse ecological questions that were previously intractable. In particular, ecologists are often interested in the demographic history of populations, information that is rarely available from historical records. Methods have been developed to infer demographic parameters from genomic data, but it is not well understood how inferred parameters compare to true population history or depend on aspects of experimental design. Here, we present and evaluate a method of SNP discovery using RNA sequencing and demographic inference using the program δaδi, which uses a diffusion approximation to the allele frequency spectrum to fit demographic models. We test these methods in a population of the checkerspot butterfly Euphydryas gillettii. This population was intentionally introduced to Gothic, Colorado in 1977 and has as experienced extreme fluctuations including bottlenecks of fewer than 25 adults, as documented by nearly annual field surveys. Using RNA sequencing of eight individuals from Colorado and eight individuals from a native population in Wyoming, we generate the first genomic resources for this system. While demographic inference is commonly used to examine ancient demography, our study demonstrates that our inexpensive, all‐in‐one approach to marker discovery and genotyping provides sufficient data to accurately infer the timing of a recent bottleneck. This demographic scenario is relevant for many species of conservation concern, few of which have sequenced genomes. Our results are remarkably insensitive to sample size or number of genomic markers, which has important implications for applying this method to other nonmodel systems.     

Assessing the degree of stratification between closely related Holstein-Friesian populations

Genomic information is an important part of the routine evaluation of dairy cattle and provides the wide availability of animals genotyped using single nucleotide polymorphism (SNP) microarrays. We analyzed 2243 Polish and 2294 German Holstein-Friesian bulls genotyped using the Illumina BovineSNP50 BeadChip. For each bull, estimated breeding values (EBVs) calculated from national routine genetic evaluation were available for production traits and for somatic cell score (SCS). Separately for each population, we estimated SNP haplotypes, pairwise linkage disequilibrium (LD), and SNP effects. The SNP genetic covariance between both populations was estimated using a bivariate mixed model. The average LD was lower in the Polish than in the German population and, with increasing genomic distance, LD decays 1.7 times more rapidly in German than in Polish cattle. The comparison of SNP allele frequencies for base populations estimated separately using Polish and German data revealed a very good agreement. The comparison of genetic effects corresponding to various window lengths defined in bp emerged a systematic pattern: regardless of the length of the compared region, few significant differences were found for production traits, while many were observed for SCS. For each trait, the German population had much higher SNP variances than the Polish population and the genetic covariance estimates were all positive. Depending on traits’ inheritance mode, the additive genetic variation can be stored in many genes following the infinitesimal model (like for SCS) or distributed between genes with high effects and the polygenic “background” (like for production traits). Accounting for those differences has implications on the prospective international genomic evaluation.     

AKT/GSK3β Signaling in Glioblastoma

Glioblastoma (GBM) is the most aggressive of primary brain tumors. Despite the progress in understanding the biology of the pathogenesis of glioma made during the past decade, the clinical outcome of patients with GBM remains still poor. Deregulation of many signaling pathways involved in growth, survival, migration and resistance to treatment has been implicated in pathogenesis of GBM. One of these pathways is phosphatidylinositol-3 kinases (PI3K)/protein kinase B (AKT)/rapamycin-sensitive mTOR-complex (mTOR) pathway, intensively studied and widely described so far. Much less attention has been paid to the role of glycogen synthase kinase 3 β (GSK3β), a target of AKT. In this review we focus on the function of AKT/GSK3β signaling in GBM.     

Glycoprotein hypersecretion alters the cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.     

Analysis of mouse Rad54 expression and its implications for homologous recombination

Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs). Important proteins in this pathway are Rad51 and Rad54. Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54. We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules. Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation. We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion. The models differ in whether Rad54 functions locally or globally. In the first model, Rad54 acts in cis relative to the site of strand invasion. Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament. Subsequently, it promotes duplex DNA unwinding. In the second model, Rad54 acts in trans relative to the site of strand invasion. Rad54 binds duplex DNA distant from the site that will be unwound. Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding.