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.     

In Vivo Interaction Proteomics Reveal a Novel p38 Mitogen-Activated Protein Kinase/Rack1 Pathway Regulating Proteostasis in Drosophila Muscle

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g., inclusion body myositis. p38 mitogen-activated protein kinase (MAPK) plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. Aging p38 MAPK mutant flies display accelerated motor function decline, concomitant with an enhanced accumulation of detergent-insoluble protein aggregates in thoracic muscles. Chemical genetic experiments suggest that p38-mediated regulation of myoproteostasis is not limited to the control of reactive oxygen species production or the protein degradation pathways but also involves upstream turnover pathways, e.g., translation. Using affinity purification and mass spectrometry, we identified Rack1 as a novel substrate of p38 MAPK in aging muscle and showed that the genetic interaction between p38b and Rack1 controls muscle aggregate formation, locomotor function, and longevity. Biochemical analyses of Rack1 in aging and stressed muscle suggest a model whereby p38 MAPK signaling causes a redistribution of Rack1 between a ribosome-bound pool and a putative translational repressor complex.     

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.     

Competing Activities of Heterotrimeric G Proteins in Drosophila Wing Maturation

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Gαs alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Gαo can specifically antagonize the Gαs activities by competing for the Gβ13F/Gγ1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.     

Post‐glacial history and introgression in Abies (Pinaceae) species of the Russian Far East inferred from both nuclear and cytoplasmic markers

Aim The main aim of the present study is to infer the post‐glacial history of Abies species from north‐east Asia and to test the hypotheses that coastal Abies populations suffered less from climatic fluctuations during Pleistocene glacial periods than their more continental counterparts, and that Sakhalin was a major area of introgression. Location Natural ranges of the fir species Abies nephrolepis, Abies sachalinensis and Abies holophylla in the Russian Far East, and of Abies gracilis, which is endemic to the Kamchatka Peninsula. Methods Nineteen populations were sampled for allozyme analysis. Seventeen of these populations were also screened for variation at two paternally inherited chloroplast DNA microsatellite loci (cpSSR) and variation at one maternally inherited mitochondrial marker (nad4‐3/4). Finally a subset of 11 populations was analysed with amplified fragment length polymorphism (AFLP). Comparisons were made with already available Abies sibirica data. For all sets of markers, we estimated genetic diversity and differentiation using an analysis of molecular variance (AMOVA). Population clustering was assessed with a Bayesian approach implemented in structure v.2.3. Results Among the three major species, A. sibirica, A. nephrolepis and A. sachalinensis, A. sachalinensis demonstrated the highest cytoplasmic and nuclear diversity and the most continental species, A. sibirica, the lowest. Both nuclear and mitochondrial DNA markers revealed the presence of a transitional zone on Sakhalin Island between A. nephrolepis and A. sachalinensis of south Sakhalin. The structure analysis delivered very clear results confirming the admixed origin of A. sachalinensis, with a genetic contribution from A. nephrolepis. No variation in cytoplasmic markers was found in A. gracilis, suggesting the occurrence of a recent bottleneck. Main conclusions There is a clear reduction of genetic diversity in Abies species from the Pacific coast into the continent. The higher diversity in A. sachalinensis could have two causes: a larger effective population size in the islands due to relatively stable climatic conditions and consequently less pronounced demographic fluctuations in population size and/or hybridization with continental and Japanese populations. Sakhalin Island is a major transitional zone for conifer species. Finally, the fir from Kamchatka, A. gracilis, should be regarded as a separate species closely related to the A. nephrolepis–A. sachalinensis complex.     

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.     

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     

Results of combined radiotherapy and hormonal treatment of prostate cancer patients with initial PSA value >40 ng/ml

AimTo evaluate the outcome of prostate cancer patients with initial PSA value >40 ng/ml.BackgroundThe outcome of prostate cancer patients with very high initial PSA value is not known and patients are frequently treated with palliative intent. We analyzed the outcome of radical combined hormonal treatment and radiotherapy in prostate cancer patients with initial PSA value >40 ng/ml.MethodsBetween January 2003 and December 2007 we treated, with curative intent, 56 patients with non-metastatic prostate cancer and initial PSA value >40 ng/ml. The treatment consisted of two months of neoadjuvant hormonal treatment (LHRH analog), radical radiotherapy (68–78 Gy, conformal technique) and an optional two-year adjuvant hormonal treatment.ResultsThe median time of follow up was 61 months. 5-Year overall survival was 90%. 5-Year biochemical disease free survival was 62%. T stage, Gleason score, PSA value, and radiotherapy dose did not significantly influence the outcome. Late genitourinal and gastrointestinal toxicity was acceptable.ConclusionRadical treatment in combination with hormonal treatment and radiotherapy can be recommended for this subgroup of prostate cancer patients with good performance status and life expectancy.