Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.     

FIMM, a database of functional molecular immunology: update 2002

FIMM database (http://sdmc.krdl.org.sg:8080/fimm) contains data relevant to functional molecular immunology, focusing on cellular immunology. It contains fully referenced data on protein antigens, major histocompatibility complex (MHC) molecules, MHC-associated peptides and relevant disease associations. FIMM has a set of search tools for extraction of information and results are presented as lists or as reports.     

Photomorphogenetic responses to UV radiation and short-term red light in lettuce seedlings

Effects of red light (R), far-red light (FR) and UV radiation on growth and greening of lettuce seedlings (Latuca sativa L., cv. Berlinskii) have been investigated. UV-B and UV-C inhibited hypocotyl elongation and stimulated cotyledonary growth. R in combination with UV-B and UV-C partly eliminated these effects, but FR increased those and reversed the R effect. Chlorophyll accumulation was inhibited by UV-B and UV-C. In comparison with cotyledonary growth, R strengthened the UV inhibitory effect, and FR reversed this effect of R. Thus, UV and phytochrome system modify the effects of each other on hypocotyl and leaf growth in lettuce seedlings depending on the level of active phytochrome formed.     

Characterization of several LINE-1 elements in Microtus kirgisorum

Several LINE-1s have been isolated and characterized from genomic DNA of the vole, Microtus kirgisorum. Blot hybridization revealed specific restriction patterns of L1 elements in vole genomes. Rehybridization of the genomic blot with a cloned 5′-end fragment revealed two major bands indicating the presence of two different L1 subfamilies. The copy numbers are estimated for different parts of M. kirgisorum L1 elements. Data also demonstrate that most vole L1 elements are truncated at the 5′-end; however, in contrast to mouse, the ORF1 copy number is higher in vole. A difference between the substitution rates of the ORF1 5′-region (approximately 330 nucleotides) and the rest of the L1 coding regions is revealed. Received: 12 January, 1999 / Accepted: 18 March, 1999     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Progression of human bone marrow stromal cells into both osteogenic and adipogenic lineages is differentially regulated by structural conformation of collagen I matrix via distinct signaling pathways

Adult human bone marrow stromal cells (BMSCs) containing or consisting of mesenchymal stem cells (MSCs) are an important source in tissue homeostasis and repair. Although many processes involved in their differentiation into diverse lineages have been deciphered, substantial inroads remain to be gained to synthesize a complete regulatory picture. The present study suggests that structural conformation of extracellular collagen I, the major organic matrix component in musculoskeletal tissues, plays, along with differentiation stimuli, a decisive role in the selection of differentiation lineage. It introduces a novel concept which proposes that structural transition of collagen I matrix regulates cell differentiation through distinct signaling pathways specific for the structural state of the matrix. Thus, on native collagen I matrix inefficient adipogenesis is p38-independent, whereas on its denatured counterpart, an efficient adipogenesis is primarily regulated by p38 kinase. Inversely, osteogenic differentiation occurs efficiently on native, but not on denatured collagen I matrix, with a low commencement threshold on the former and a substantially higher one on the latter. Osteogenesis on collagen I matrices in both structural conformations is fully dependent on ERK. However, whereas on native collagen I matrix osteogenic differentiation is Hsp90-dependent, on denatured collagen I matrix it is Hsp90-independent. The matrix conformation-mediated regulation appears to be one of the mechanisms determining differentiation lineage of BMSCs. It allows a novel interpretation of the bone remodeling cycle, explains the marked physiological aging-related adipogenic shift in musculoskeletal tissues, and can be a principal contributor to adipogenic shift seen in a number of clinical disorders.     

Study of non-consumptive mortality of Crustacean zooplankton in a Siberian reservoir using staining for live/dead sorting and sediment traps

We studied non-consumptive (non-predatory) mortality of Daphnia and Cyclops vicinus during four sampling seasons. Mortality estimations were based on live/dead sorting using special staining and measurements of sedimentation rates for dead individuals, depended on wind speed. Original equations were used for calculations. The estimated specific non-consumptive mortality never had biologically senseless negative values, which were often obtained on the basis of the other ways of mortality estimations, and was in a good agreement with other components of population dynamics. As found, the non-consumptive mortality was the important, often the determinant component of the zooplankton population dynamics.     

Changes in the starvation response through covalent cell attachment

Covalent attachment of Candida utilis cells, possibly simulating natural microbial immobilizations, stimulated stable and significant enhancement of extracellular production of alkaline protease, specifically induced by four different starvation conditions. The enzyme analysis confirmed the identity of the proteases released under all conditions of starvation and no parallel production of other proteolytic enzyme. The enhancement phenomenon as a uniform and stable effect of the whole cell immobilization is discussed in relation to the effect of multipoint, cell-solid surface contact, potentially bringing positive modulations of complex, cellular functions.     

Development of seawater adaptation in pink salmon,Oncorhynchus gorbuscha,during downstream migration: relationships to temperature and residual yolk

Synopsis Seawater adaptability of wild pink salmon, Oncorhynchus gorbuscha, alevins was investigated using 96 h LC50 test, muscle sodium level and weight loss tests. Five temperature (2, 6, 9, 13 and 16°C) and two yolk groups were analysed during the beginning, the middle and the end of downstream migration. Both temperature and amount of residual yolk affected seawater adaptability. The computation of muscle sodium levels and weight loss after a short term (3–12 h) seawater challenge test may be a convenient method to estimate the seawater adaptability of fish too small to allow analysis of blood sodium concentration.