Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties

Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.     

The genes of two G-proteins involved in protein transport in Pichia pastoris

Members of the Rab protein family play essential roles in vesicle fusion during protein secretion and represent highly conserved GTP binding proteins. The Saccharomyces cerevisiae Sec4p and Ypt1p, promoting vesicle fusion at the plasma membrane and in ER-Golgi transport, respectively, are among the best characterised yeast members. We have here cloned the Pichia pastoris SEC4 homologue using a S. cerevisiae SEC4 probe. In addition we isolated a crosshybridising clone encoding another Rab-/Ypt-like protein. The deduced full-length PpSec4p comprises 204 amino acid residues with an over all identity of 64% to the Sec4p from S. cerevisiae and 72% to the Candida albicans Sec4p. The YPT-like gene encodes a 216 amino acid residue protein showing highest similarity to the S. cerevisiae Ypt10p and Ypt53p. Both PpSec4p and the Ypt-like protein carry a -Cys-Cys C-terminus, indicating that these proteins are targets for geranyl-geranylation by a type II prenyltransferase.     

Selection of cell‐type specific antibodies on tissue‐sections using phage display

With the advent of modern technologies enabling single cell analysis, it has become clear that small sub‐populations of cells or even single cells can drive the phenotypic appearance of tissue, both diseased and normal. Nucleic acid based technologies allowing single cell analysis has been faster to mature, while technologies aimed at analysing the proteome at a single cell level is still lacking behind, especially technologies which allow single cell analysis in tissue. Introducing methods, that allows such analysis, will pave the way for discovering new biomarkers with more clinical relevance, as these may be unique for microenvironments only present in tissue and will avoid artifacts introduced by in vitro studies. Here, we introduce a technology enabling biomarker identification on small sub‐populations of cells within a tissue section. Phage antibody libraries are applied to the tissue sections, followed by washing to remove non‐bound phage particles. To eliminate phage antibodies binding to antigens ubiquitously expressed and retrieve phage antibodies binding specifically to antigens expressed by the sub‐population of cells, the area of interest is protected by a ‘shadow stick’. The phage antibodies on the remaining areas on the slide are exposed to UV light, which introduces cross‐links in the phage genome, thus rendering them non‐replicable. In this work we applied the technology, guided by CD31 expressing endothelial cells, to isolate recombinant antibodies specifically binding biomarkers expressed either by the cell or in the microenvironment surrounding the endothelial cell.     

Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the induced neurodegeneration, we have developed standardized protocols, including--a) densitometric measurements of the cellular uptake of propidium iodide (PI), --b) histological staining by Flouro-Jade, --c) lactate dehydrogenase (LDH) release to the culture medium, --d) immunostaining for microtubulin-associated protein 2, and --e) general and specific neuronal and glial cell stains. The results show good correlation between the different markers, and are in accordance with results obtained in vivo. Examples presented in this review will focus on the use of PI uptake to monitor the excitotoxic effects of --a) KA and AMPA (and NMDA) in hippocampal slice cultures, and --b) KA and AMPA in corticostriatal slice cocultures, with demonstration of differentiated neuroprotective effects of NBQX in relation to cortex and striatum and KA and AMPA. A second set of studies include modulation of hippocampal KA-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection.     

Constant, cycling, hot and cold thermal environments: strong effects on mean viability but not on genetic estimates

It has frequently been suggested that trait heritabilities are environmentally sensitive, and there are genetic trade-offs between tolerating different environments such as hot and cold or constant and fluctuating temperatures. Future climate predictions suggest an increase in both temperatures and their fluctuations. How species will respond to these changes is uncertain, particularly as there is a lack of studies which compare genetic performances in constant vs. fluctuating environments. In this study, we used a nested full-sib/half-sib breeding design to examine how the genetic variances and heritabilities of egg-to-adult viability differ at high and low temperatures with and without daily fluctuations in temperatures using Drosophila melanogaster as a model organism. Although egg-to-adult viability was clearly sensitive to developmental temperatures, heritabilities were not particularly sensitive to developmental temperatures. Moreover, we found that egg-to-adult viabilities at different developmental temperatures were positively correlated, suggesting a common genetic background for egg-to-adult viability at different temperatures. Finding both a uniform genetic background coupled with rather low heritabilities insensitive to temperatures, our results suggest evolutionary responses are unlikely to be limited by temperature effects on genetic parameters or negative genetic correlations, but by the direct effects of stressful temperatures on egg-to-adult viability accompanied with low heritabilities.     

A high-throughput approach for measuring temporal changes in the interactome

Interactomes are often measured using affinity purification-mass spectrometry (AP-MS) or yeast two-hybrid approaches, but these methods do not provide stoichiometric or temporal information. We combine quantitative proteomics and size-exclusion chromatography to map 291 coeluting complexes. This method allows mapping of an interactome to the same depth and accuracy as AP-MS with less work and without overexpression or tagging. The use of triplex labeling enables monitoring of interactome rearrangements.     

Phenotypic characterization of cells participating in transport of prion protein aggregates across the intestinal mucosa of sheep

The oral route is considered to be the main entry site of several transmissible spongiform encephalopathies or prion diseases of animals and man. Following natural and experimental oral exposure to scrapie, sheep first accumulate disease associated prion protein (PrP^d) in Peyer’s patch (PP) lymphoid follicles. In this study, recombinant ovine prion protein (rPrP) was inoculated into gut loops of young lambs and the transportation across the intestinal wall studied. In particular, the immunohistochemical phenotypes of cells bearing the inoculated prion protein were investigated. The rPrP was shown to be transported across the villi of the gut, into the lacteals and submucosal lymphatics, mimicking the transport route of PrP^d from scrapie brain inoculum observed in a previous intestinal loop experiment. The cells bearing the inoculated rPrP were mainly mononuclear cells, and multicolor immunofluorescence procedures were used to show that the rPrP bearing cells were professional antigen presenting cells expressing Major histocompatibility complex II (MHCII). In addition, the rPrP bearing cells labeled with CD205, CD11b and the macrophage marker CD68, and not with the dendritic cell markers CD11c and CD209. Others have reported that cells expressing CD205 and CD11b in the absence of CD11c have been shown to induce T cell tolerance or regulatory T cells. Based on this association, it was speculated that the rPrP and by extension PrP^d and scrapie infective material may exploit the physiological process of macromolecular uptake across the gut, and that this route of entry may have implications for immune surveillance.     

Eurocin,a New Fungal Defensin: STRUCTURE,LIPID BINDING,AND ITS MODE OF ACTION*

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.