Distribution of alpha-amylase isozymes among the varieties of winter wheat in Russia and Ukraine

A catalog of winter wheat varieties bred in Russia and Ukraine for alpha-amylase isozymes is presented. Among them, 11 phenotypic classes were found. It was established that the observed differences in the frequency of specific phenotypes for alpha-amylase in the culture of winter wheat depended on the geographical origin. The percentage of the most widespread cultural phenotype, designated as AbCde, decreased from the south (45°–46° N) to the north (49°–50° N) by 31.0%. At the same time, the frequency of the abCde variant increased by 25.0%. The distribution of the Abcde phenotype changed significantly from 20.7% in the west (30o36′ E) to 0% in the east (40o18′ E). The observed territorial dynamics for alpha-amylase isoenzymes may indicate the adaptive value of the identified phenotypes of common winter wheat for the weather and climatic conditions of the region of their origin.     

Protein markers for identification of different species and varieties of cotton

Reference electrophoretic spectra that allow compiling electrophoretic formulas of certain cotton species and varieties were obtained on the basis of analysis of the electrophoretic spectrum of water-soluble and barely soluble proteins of seeds of diploid cotton species of genomic group A (Gossypium arboretum var. indicum, G. arboreum ssp. obtusifolum, G. herbaceum ssp. africanum, and G. herbaceum Harga), group C (G. australe, G. bickii, G. nelsone, and G. sturtianum), group D (G. davidsonii. G. harknessii. G. klotzschianum, G. raimondii, G. thurberi, and G. trilobum), and amphidiploid species of group AD (G. mustelinum, G. hirsutum ssp. palmeri, G. tricuspidatum Bagota, G. tricuspidatum Mari Galanta, G. barbadense L., and G. hirsutum L.).     

Protein markers for identification of different species and varieties of cotton

Reference electrophoretic spectra that allow compiling electrophoretic formulas of certain cotton species and varieties were obtained on the basis of analysis of the electrophoretic spectrum of water-soluble and barely soluble proteins of seeds of diploid cotton species of genomic group A (Gossypium arboreum var. indicum, G. arboreum ssp. obtusifolum, G. herbaceum ssp. africanum, and G. herbaceum Harga), group C (G. australe, G. bickii, G. nelsone, and G. sturtianum), group D (G. davidsonii, G. harknessii, G. klotzschianum, G. raimondii, G. thurberi, and G. trilobum), and amphidiploid species of group AD (G. mustelinum, G. hirsutum ssp. palmeri, G. tricuspidatum Bagota, G. tricuspidatum Mari Galanta, G. barbadense L., and G. hirsutum L.)     

出芽短梗霉原生质体再生变异菌株的同工酶及可溶性蛋白的研究

用聚丙烯酰胺凝胶电泳技术对出芽短梗霉的亲本菌株及其４株原生质体再生菌株的脂酶、谷氨酸脱氢酶、细胞色素氧化酶和可溶性蛋白进行了比较。发现各变异菌株之间,变异菌株与亲本菌株之间的谱带均有差异,原生质体再生菌株的变异涉及酶蛋白的改变。     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.     

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.     

Use of bone morphogenetic proteins in mesenchymal stem cell stimulation of cartilage and bone repair

The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell(MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted celltype lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair.     

Computational identification of proteins for selectivity assays

At the stage of optimization of a chemical series the compounds are normally assayed for binding or inhibition on the target protein as well as on several proteins from a selectivity panel. These proteins are normally identified on the basis of sequence homology to the target protein. Experimental selectivity data are also taken into account if available. Cases when a nonhomologous protein has a significant affinity to the compound series are going to be missed if the selectivity panel is identified by homology. Experimental data is usually either unavailable or limited to a small fraction of proteins that should be considered. We have developed a computational method of identification of selectivity panel proteins. It is based on the evaluation of binding site similarity to the target protein using docking scores of target-selected molecular probes. These probes are obtained by docking a large library of drug-like compounds to the target protein followed by selecting a diverse subset from the best virtual binders. Docking scores of these probes to other proteins measure binding site similarity to the target. Because the method does not require prior knowledge of either affinities or structures of inhibitors for the target, it can be applied to any protein with known 3D structure. Validation of the method includes rediscovery of nonhomologous proteins that bind common ligands: estradiol, tamoxifen, and riboflavin. Given 3D structures, the method can effectively discriminate proteins with similar binding sites from random proteins independent of sequence homology.     

Multiple structure alignment and consensus identification for proteins

Background  

An algorithm is presented to compute a multiple structure alignment for a set of proteins and to generate a consensus (pseudo) protein which captures common substructures present in the given proteins. The algorithm represents each protein as a sequence of triples of coordinates of the alpha-carbon atoms along the backbone. It then computes iteratively a sequence of transformation matrices (i.e., translations and rotations) to align the proteins in space and generate the consensus. The algorithm is a heuristic in that it computes an approximation to the optimal alignment that minimizes the sum of the pairwise distances between the consensus and the transformed proteins.     

Use of isoenzymes for the identification of apricot cultivars

Isozyme polymorphism was used for identification of apricot cultivars. 34 cultivars of 38 studied ones had unique genotype profiles for allelic composition of the AAT-1, EST-1, EST-2, SOD-1, LAP-1, PX-2 loci and AMY phenotypes. All regionized and being under State sort testing cultivars of hybridous origin were differentiated. Cultivars of clonal selection were identified regarding cultivars type.     

Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins

Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.     

Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.     

A target-specific approach for the identification of tyrosine-sulfated hemostatic proteins

A simple methodology for the identification of hemostatic proteins that are subjected to posttranslational tyrosine sulfation was developed. The procedure involves sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm, followed by [³⁵S]sulfate labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [³⁵S]sulfate-labeled hemostatic proteins, and tyrosine O-[³⁵S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins—protein S, prekallikrein, and plasminogen—were identified. Such a target-specific approach will allow investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understanding of the functional role of posttranslational tyrosine sulfation.