Putative high mobility group (HMG) non-histone chromosomal proteins from wheat germ. Isolation, characterisation and partial sequence analysis

Four proteins have been isolated from wheat germ by methods analogous to those used to isolate HMG proteins from animal tissue. All four proteins have been shown to be chromosomal in origin. Although amino acid analyses show that three of these proteins have compositions similar to those of the mammalian HMG proteins, N-terminal sequence analyses of these proteins show an absence of sequence homology with any of the mammalian HMG proteins.     

Cerebrospinal Fluid Proteins Studied by Two-Dimensional Gel Electrophoresis and Immunoblotting Technique

The proteins of lumbar CSF have been investigated by two-dimensional gel electrophoresis, and their patterns have been compared with the corresponding serum protein patterns. Serum proteins in CSF have been identified by electroblotting and immunoreaction with antiserum against total human serum proteins. Proteins derived from brain have been identified with antiserum against human brain proteins. The most prominent CSF protein group has been identified as a multiple form of apolipoprotein E. The correct position of the glial fibrillary acidic protein has also been determined. The prefractionation of CSF proteins by size exclusion chromatography or by affinity chromatography followed by two-dimensional electrophoresis has facilitated the detection of trace components in CSF and the corresponding serum.     

Heterogeneity of nuclear proteins from HeLa cells specifically binding to the Alu sequence

Nuclear proteins from HeLa cells specifically binding to the Alu-repeat cloned in the plasmid Blur8 have been studied. 0.35 M nuclear extract proteins have been separated on DEAE-cellulose. The presence of DNA-binding proteins has been found in all fractions by the technique of DNA-binding on nitrocellulose filters. The labelled restricted DNA of the plasmid Blur8 was incubated with the proteins of different fractions with the subsequent identification of specific Alu-protein complexes in polyacrylamide gel at low ionic strength. At least two proteins have been found to have the different affinity to Alu-repeat. Various functions of Alu-repeats and the possibility of their participation in the initiation of DNA replication are discussed.     

The effect of cytoplasmic proteins on the reaggregation of dissociated ghost membranes of Bacillus megaterium KM

The effect of cytoplasmic proteins on the reassociation of membrame proteins and lipids which have been solubilized in sodium dodecyl sulfate and urea has been investigated. The cytoplasmic proteins have been found to inhibit the reassociation of the membrane proteins. Moreover, approximately 15% of the cytoplasmic proteins co-aggregate with the membrane components after removal of the sodium dodecyl sulfate and urea.     

Studies on the proteins of Poecilocera picta. I-changes taking place in the haemolymph protein during moulting

The haemolymph proteins of the 6th nymph of P. picta were fractioned by the polyacrylamide gel disc electrophoresis. A total of seven proteins fractions have been detected from the haemolymph. The chemical nature of different protein fractions have been examined by histochemical methods. The changes taking place in the cuticle and epidermal cells have been examined during the transformation of 6th nymph into adult. The fat body proteins have been electrophoretically fractioned and the changes in the concentration of different protein fractions have been examined. It is suggested that the protein fraction 3 of the haemolymph is utilized in the formation of new cuticle. It is concluded by the histochemical observations that proteins of the band 3 are synthesized in the fat body.     

The role of stallion seminal proteins in fertilisation

Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitation and gamete interaction. The major equine seminal plasma proteins belong to three protein classes, which contain widely occurring protein modules. Fn-2 type proteins are characterised by two or four tandemly arranged Fn-2 modules and have been implicated in the modulation of sperm capacitation. Multiple members of the cysteine-rich secretory proteins (CRISP) have been identified in the male genital tract of a number of species. CRISP proteins have been shown to be involved in various functions related to sperm-oocyte fusion, innate host defense function and ion channel blockage. Spermadhesins occur only in ungulate species. Their carbohydrate- and zona pellucida-binding properties would suggest a role of these proteins in gamete recognition. The major proteins of equine seminal plasma have been isolated and characterised regarding their expression along the male genital tract, protein structure and their functions.     

Proteomic analysis of mitochondria from the Bos taurus heart. III. Identification of mitochondrial inner memrane proteins

We have conducted a research of mitochindrial internal membrane proteins. This fraction has been received in the form of submitochondrial particles (SMP). SMP have been processed by trypsinum, and the received peptides have been separated from so-called "smoothfaced vesicles". "Smoothfaced vesicles" were blasted, proteinse and peptides were processed by cyanogen bromide and trypsinum. We have received two groups of tryptic peptides and analyzed them separately with the help of proteomic methods, such as chromatography, mass spectrometry and protein identification in different databases. To identify more proteins and find minor components of mitochindrial proteome we have considered possible non-specific fragmentation of proteins. 298 proteins have been identified, we also have conducted the analisys of their functions and cell localization.     

Regulatory GTP-binding proteins: emerging concepts on their role in cell function

The last few years have evidenced a tremendous expansion in our appreciation of the role of regulatory GTP-binding proteins in cellular activation. The availability of cholera and pertussis toxins to detect G proteins as well as methodological advances in the study of cellular function has afforded the opportunity to examine G protein participation in many cellular events. Regulation of adenylyl cyclase and cyclic GMP phosphodiesterase by G proteins has been demonstrated. Phosphatidylinositol-4,5-biphosphate specific phospholipase C activity appears to be subject to G protein control. G proteins regulate inward K+ and Ca2+ channels through a mechanism which may be independent of effects on the above mentioned enzymes. Certainly, the number of G proteins which have been identified from sequencing of complementary DNA affords the potential for G protein involvement in many cellular events. Only three G proteins have however been isolated and functionally characterized, Gs, Gi and transducin. Whether all the functions of these proteins have been identified remains to be seen.     

Sowing the seeds of success: pharmaceutical proteins from plants

Among the many plant-based production systems that have been developed for pharmaceutical proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume and in a stable environment in which they are protected from degradation. Several seed crops, including cereals, grain legumes and oilseeds, have been explored as production platforms, and the first commercial products -- all technical proteins and enzymes -- have already reached the market. Recent studies have explored the use of seeds for the production of pharmaceutical proteins, particularly replacement human proteins, recombinant antibodies and (oral) vaccines.     

Sedimentation equilibrium of proteins in density gradients.

The technique of sedimentation equilibrium in density gradients in the analytical ultracentrifuge has been applied to the study of proteins. A variety of effects and procedures including the use of density marker beads, the effects of pressure on buoyant density and pH, and the calculation of compositional density gradient proportionality constants and density--refractive index relations have been developed. The buoyant densities of twenty-four proteins have been measured and hydration values computed. The buoyant titrations of six proteins have been measured. These data have been interpreted in terms of the buoyant titrations which have been obtained for six ionizable homopolypeptides, five copolypeptides, two non-ionizable homopolypeptides and three chemically modified proteins. Spectropolarimetry and potentiometric titrations were employed to further interpret these data. Approximate values for dissociation constants, numbers of ionizable residues, and the nature of ions bound or dissociated upon ionization have been obtained. The relation between potentiometric and buoyant titrations and the use of density gradient centrifugation as a probe for protein structure have been explored.     

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Repetitive proline-rich proteins in the extracellular matrix of the plant cell

Several classes of hydroxyproline-rich proteins have been found in the cell walls of plants. Monomeric forms of the proteins can be solubilized from the walls, but the majority of the proteins are insolubilized by as yet unidentified crosslinks. The proteins have repeated sequences and are often rich in basic amino acids. The repeat proline-rich region may be serving to generate an elongated rod-like structure while the regularly spaced lysines probably interact with the acidic pectins. Several of the genes coding for these proteins have been isolated, and their expression has been found in some cases to be tissue specific and in others inducible by hormones and various types of stress.     

Topography of the total protein population from cultured cells upon fractionation by chemical extractions

Chemical extractions are proposed as a major tool for a fractionation of cellular proteins. As a model system, proteins from cultured hamster lens cells have been divided by independent extractions into seven subcellular fractions, corresponding to water-soluble proteins and the proteins from membranes, microfilaments (and other deoxycholate-soluble proteins), intermediate filaments, microtubules, polysomes and nuclei respectively. The latter two fractions have been subfractionated yielding ribosomal proteins, the elongation and initiation factors of the protein-synthesis machinery, chromatin proteins and non-chromatin proteins. The protein compositions of the fractions have been analyzed by one-dimensional and two-dimensional gel electrophoresis. This resulted in an almost complete topography of the proteins detected on two-dimensional gels of total-cell lysates. Comparison of two-dimensional patterns of proteins from the total-cell lysate and proteins from hamster erythrocytes or from liver, muscle or brain tissue showed that the different cell types have only few proteins in common. Two proteins are common to all of these cell types, namely actin and a 68-kDa protein. The latter protein was, like actin, vimentin and the tubulin subunits, also present in most cell fractions. Evidence is presented that this protein is identical to a 68-kDa heat-shock protein.     

High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

Cellular retinol-binding proteins of the mucous membrane of the intermediate zone of the glandular stomach in chickens

The cellular retinol-binding proteins with the molecular weight of 14 and 53.5 kDa have been isolated from the intermediate zone mucosa cells of the glandular stomach in chickens. No substantial differences in the amino acid composition of the investigated proteins have been found. The possible functional role of the isolated cellular retinol-binding proteins is discussed.     

Design and discovery of metamorphic proteins

Metamorphic proteins are single amino acid sequences that reversibly interconvert between multiple, dramatically different native structures, often with distinct functions. Since the discovery of the first metamorphic proteins in the early 2000s, several additional metamorphic proteins have been identified, and it was suggested that up to 4% of proteins in the PDB may switch folds. Metamorphic proteins have been found to share common features such as marginal thermostability and inconsistencies in predicted secondary structures. Outstanding challenges in the field include the search for more metamorphic proteins and the design of new proteins that switch folds. Identification of novel metamorphic proteins in nature will improve therapeutic targeting of fold-switching proteins involved in human pathology and will enhance the design of protein-based therapies. Designed fold switching proteins have applications as biosensors, molecular switches, molecular machines, and self-assembling systems.     

Centromere and kinetochore structure.

Recent studies have begun to yield some insight into the structural and regulatory components of centromeres, and new assays have been developed that promise to be of use in advancing our understanding of centromere structure and function. In the budding yeast Saccharomyces cerevisiae new proteins that are required for centromere function have been identified and an in vitro microtubule-binding assay that should assist in dissecting the process of centromere microtubule attachment has been developed. The centromere-specific DNA sequences in the fission yeast Schizosaccharomyces pombe have been identified and partially characterized. In addition, several mammalian centromere proteins have been further characterized, and localization and inhibition studies suggest roles for these proteins in the regulation and assembly of a functional kinetochore.