Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.     

Beyond 100 genomes

By the end of 2002, we witnessed the landmark submission of the 100th complete genome sequence in the databases. An overview of these genomes reveals certain interesting trends and provides valuable insights into possible future developments.     

Fas is not essential for lamina propria T lymphocyte homeostasis

IL-2 receptor alpha-deficient (IL2Ralpha-/-) mice spontaneously accumulate vast numbers of intestinal lamina propria (LP) T cells and develop bowel inflammation. The accumulation of T cells in IL2Ralpha-/- mice is thought to result, in part, from defective Fas-induced cell death. To understand the role of cell proliferation and death in regulating LP T cells in IL2Ralpha-/- mice, we have directly examined the proliferation and Fas sensitivity of wild-type, lpr/lpr, and IL2Ralpha-/- LP T cells. In wild-type mice, 5'-bromodeoxyuridine (BrdU) labeling and Fas susceptibility are greatest in CD44Hi LP T cells. Fas-deficient lpr/lpr mice have normal total numbers of LP T cells, despite an increased proportion of BrdU+ T cells. By contrast, IL2Ralpha-/- mice possess increased total numbers of LP T cells, despite normal proportions of BrdU+ LP T cells. Finally, wild-type and IL2Ralpha-/- LP T cells are equivalently Fas sensitive. These results demonstrate that LP T cells proliferate and are Fas-sensitive cells. IL2Ralpha-/- mice accumulate a large number of these Fas-sensitive LP T cells and clearly differ from Fas-deficient lpr/lpr mice in this regard. Thus our studies reveal that Fas is dispensable for LP T cell homeostasis and suggest that the intestinal inflammation observed in IL2Ralpha-/- mice is independent of defective Fas-induced cell death.     

The synthesis of an analogue of the locust CRF-like diuretic peptide, and the biological activities of this and some C-terminal fragments

The synthesis is described of an analogue of the locust CRF-like diuretic peptide in which methionine in positions 1,3, and 13 is replaced by isosteric methyl-homoserine residues. This analogue has been tested for biological activity on Malpighian tubules in vitro, and feeding behavior in vivo. It is highly active in stimulating fluid secretion and accumulation of cAMP in tubules, and on increasing the latency to feed and reducing meal duration. A 15 residue fragment from the C-terminus of the CRF-like peptide, Locmi-DP(32-46), is fully active in the feeding assay, but has only weak ability to stimulate the accumulation of cAMP in tubules. Two smaller fragments, Locmi-DP(32-37) and Locmi-DP(41-46), were tested but neither had consistent biological activity in any of the assays used here. None of the peptides tested have any substantive activity in increasing cGMP in tubules.     

From the mountains to the sea: assemblage structure and dynamics in Chironomidae (Insecta: Diptera) in the Clyde River estuarine gradient, New South Wales, south-eastern Australia

Abstract  Chironomidae (Insecta: Diptera: non-biting midges) were surveyed at five shallow riffle stations along the estuarine gradient of the Clyde River, southern New South Wales (35°45'S, 150°15'E). Benthic populations were sampled seasonally between April 2001 and January 2002, between an uppermost fully fresh station and 7 km south of the tidal limit at Shallow Crossing, encompassing a 23 km stretch. Biological structure and integrity of chironomid assemblages, which are essentially unknown in eastern Australia's estuarine environments, were elucidated. Assemblages were diverse: from 5732 chironomid larvae, 45 species belonging to four subfamilies were identified from riffles. All chironomid assemblages were strongly structured and non-random with respect to spatial position along the salinity gradient although relatively random with respect to temporal shifts between the five seasonal samples. Generally, the salinity gradient had strong effects on assemblage composition but no discrete brackish fauna was identifiable, and the abundance of many species declined gradually with distance from the freshest station. Dominant taxa in the brackish zone were Parakiefferiella ' variegatus ' and two species of Cladotanytarsus . Notably, the little-known Semiocladius crassipennis Skuse (Orthocladiinae) was abundant at the most marine-influenced station. Taxa present exclusively in freshwaters included several Tanypodinae notably absent from sites below Shallow Crossing at salinities normally tolerated in athalassic waters. Other species restricted to freshwaters included Nanocladius sp., Demicryptochironomus (Irmakia) sp., Polypedilum vespertinus (Skuse), Zavrelliella fuscoguttata (Kieffer), Riethia stictoptera (Kieffer) and Podonomopsis sp.     

Widespread Changes in White Matter Microstructure after a Day of Waking and Sleep Deprivation

BackgroundElucidating the neurobiological effects of sleep and waking remains an important goal of the neurosciences. Recently, animal studies indicated that sleep is important for cell membrane and myelin maintenance in the brain and that these structures are particularly susceptible to insufficient sleep. Here, we tested the hypothesis that a day of waking and sleep deprivation would be associated with changes in diffusion tensor imaging (DTI) indices of white matter microstructure sensitive to axonal membrane and myelin alterations.MethodsTwenty-one healthy adult males underwent DTI in the morning [7:30AM; time point (TP)1], after 14 hours of waking (TP2), and then after another 9 hours of waking (TP3). Whole brain voxel-wise analysis was performed with tract based spatial statistics.ResultsA day of waking was associated with widespread increases in white matter fractional anisotropy, which were mainly driven by radial diffusivity reductions, and sleep deprivation was associated with widespread fractional anisotropy decreases, which were mainly explained by reductions in axial diffusivity. In addition, larger decreases in axial diffusivity after sleep deprivation were associated with greater sleepiness. All DTI changes remained significant after adjusting for hydration measures.ConclusionsThis is the first DTI study of sleep deprivation in humans. Although previous studies have observed localized changes in DTI indices of cerebral microstructure over the course of a few hours, further studies are needed to confirm widespread DTI changes within hours of waking and to clarify whether such changes in white matter microstructure serve as neurobiological substrates of sleepiness.     

Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage

To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.     

Non-Steroidal Anti-Inflammatory Drug Use and Genomic DNA Methylation in Blood

Background

Non-steroidal anti-inflammatory drug (NSAID) use is associated with decreased risk of some cancers. NSAID use modulates the epigenetic profile of normal colonic epithelium and may reduce risk of colon cancer through this pathway; however, the effect of NSAID use on the DNA methylation profile of other tissues including whole blood has not yet been examined.

Findings

Using the Sister Study cohort, we examined the association between NSAID usage and whole genome methylation patterns in blood DNA. Blood DNA methylation status across 27,589 CpG sites was evaluated for 871 women using the Illumina Infinium HumanMethylation27 Beadchip, and in a non-overlapping replication sample of 187 women at 485,512 CpG sites using the Infinium HumanMethylation450 Beadchip. We identified a number of CpG sites that were differentially methylated in regular, long-term users of NSAIDs in the discovery group, but none of these sites were statistically significant in our replication group.

Conclusions

We found no replicable methylation differences in blood related to NSAID usage. If NSAID use does effect blood DNA methylation patterns, differences are likely small.     

Independent Association of Postdoctoral Training with Subsequent Careers in Cancer Prevention

The purpose of this study was to examine the career paths of alumni from the National Cancer Institute (NCI) Cancer Prevention Fellowship Program (CPFP), a structured in-house postdoctoral training program of 3–4 years duration, and specifically what proportion of the alumni were currently performing cancer prevention-related activities. The analyses here included 119 CPFP alumni and 85 unsuccessful CPFP applicants, all of whom completed postdoctoral training between 1987–2011 and are currently employed. Postdoctoral training experiences and current career outcomes data were collected via online surveys. Differences between groups were assessed using chi-square and Fisher’s exact test p-values and subsequent regression analyses adjusted for differences between the groups. Compared to 15.3% of unsuccessful CPFP applicants, 52.1% of CPFP alumni (odds ratio [OR] = 4.99, 95% confidence interval [95% CI): 1.91–13.0) were currently spending the majority of their time working in cancer prevention. Among those doing any cancer prevention-focused work, 54.3% of CPFP alumni spent the majority of their time performing cancer prevention research activities when compared to 25.5% of unsuccessful applicants (OR = 4.26, 95% CI: 1.38–13.2). In addition to the independent effect of the NCI CPFP, scientific discipline, and employment sector were also associated with currently working in cancer prevention and involvement in cancer prevention research-related activities. These results from a structured postdoctoral training program are relevant not only to the cancer prevention community but also to those interested in evaluating alignment of postdoctoral training programs with available and desired career paths more broadly.