Polypeptide Content Research Articles

Regulation of glutamine synthetase expression in sunflower cells exposed to salt and osmotic stress

Sunflower ( Helianthus annuus L. cv. SH222) plants and calluses were exposed to 100 mM NaCl and to osmotic stress induced by polyethylene glycol 6000 (PEG 6000; 50 g/l). Salt and osmotic stresses increased cytosolic glutamine synthetase (GS1) mRNA and the corresponding polypeptides in plants. In calluses, GS1 mRNA and polypeptide contents increased in response to salt stress. Determination of glutamine synthetase isoenzyme activities showed increases of GS1 activity in NaCl-stressed cells (increases of 3.5; 6.2 and 1.3 times, respectively in leaves, roots and calluses) and in PEG-stressed plants (increases of 4.0 and 6.9 times in leaves and roots, respectively), while plastidial glutamine synthetase (GS2) activity decreased in NaCl-stressed leaves. This increase of GS1 expression was accompanied by an increase of ammonium levels in stressed leaves. This report shows that NaCl and osmotic stresses induced GS1 expression by increasing GS1 mRNA and polypeptide leading to increased enzymatic GS1 activity. These data confirm previous suggestions about the increase of GS1 expression in senescent cells and support the role of this isoenzyme on nitrogen mobilization in plant cells.

Change in Lectin Specificity of Winter Wheat Seedlings in the Course of Infection with Mycoplasms

The activity of soluble lectins in leaves and roots of seedlings of winter wheat (Triticum aestivum L.) cultivar Mironovskaya 808 increased 1 day and 2 days, respectively, after infection with the mycoplasma Acholeplasma laidlawii 118. Analysis of acid-soluble proteins of wheat leaves by PAGE revealed the appearance of 22- and 20-kDa polypeptides, the disappearance of a 14-kDa polypeptide, and an increase in the content of polypeptides with molecular weights of 76, 48, 25, and 18 kDa. The 18-kDa polypeptide is a subunit of wheat germ agglutinin. A change in the activity of lectins may be a nonspecific response of plants to infection with the pathogen.

Investigation of foam‐active polypeptides during beer fermentation

AbstractDuring ale fermentation there was an accumulation of total and hydrophobic polypeptides in the foam relative to the wort. Comparisons were made not only of the total and hydrophobic polypeptide contents but also of the molecular weights of these polypeptides present in wort, partially fermented wort and its concomitant foam. Wort, fermented wort and foam fractions had very similar polypeptide compositions with a major group having molecular weights of 40–43 kDa. Material of molecular weight in the range of 5–17 kDa and at 66 kDa was also detected. The polypeptides accumulated in foam displayed both hydrophobic and non‐hydrophobic character. The presence of yeast polypeptides in foam was confirmed. Comparison was also made between the fermentations of 10°Plato and 15°Plato wort. The results of the work may contribute to a better understanding of the mechanism of foam formation during beer fermentation, leading to reduced foaming and enabling an increase in the working capacities of fermenters. Copyright © 2004 Society of Chemical Industry

Comparisons of force fields for proteins by generalized-ensemble simulations

Secondary structural characteristics of six commonly used force fields for protein systems developed by different research groups have been compared. We performed molecular dynamics simulations of an α-helical polypeptide and a β-hairpin polypeptide with explicit water molecules. Two generalized-ensemble algorithms, replica-exchange multicanonical algorithm and multicanonical replica-exchange method, for efficient sampling of configurational space have been employed. Comparisons of the secondary structure content of polypeptides for different force fields highlighted differences of their structural tendency. The results imply that α-helix is favored for AMBER94 and AMBER99 and that β-hairpin is favored for GROMOS96, while CHARMM22, AMBER96, and OPLS-AA/L have intermediate tendency.

Proteomic analysis of the oxygen-evolving complex of photosystem II under biotec stress: Studies on Nicotiana benthamiana infected with tobamoviruses.

We have previously shown that tobamovirus infection induces an inhibition of photosystem II electron transport, disturbing the oxygen-evolving complex (OEC). In the infected plants, the OEC polypeptide pattern was modified when compared to healthy plants, the levels of the PsbP and PsbQ extrinsic proteins being lowered to different extents. In this work we have further investigated by two-dimensional polyacrylamide gel electrophoresis (2-DE) the changes on the OEC protein pattern of thylakoid membranes isolated from Nicotiana benthamiana Domin plants infected with the Spanish strain of pepper mild mottle virus. When the thylakoid membranes from healthy plants were analyzed for the presence of PsbO and PsbP proteins by 2-DE (pI range 4-7) and further immunoassayed by using specific-antisera against these two proteins, it was observed that four polypeptides cross-reacted with each antiserum. These data, along with the N-terminal amino acid sequence determined for the eight polypeptides, indicate that the N. benthamiana PsbO and PsbP proteins correspond to protein families. In the silver-stained 2-DE gels of thylakoid membranes isolated at different days postinoculation from virus-infected plants, it was observed that the content of PsbP polypeptides decreased dramatically with respect to those of PsbO, during the progress of the infection. Interestingly, there was a differential decrease of the different PsbP proteins, indicative of a distinct regulation of their expression.

Typical changes of erythrocytes in chronic inflammation.

Structural and metabolic status and function of erythrocytes were studied in patients with chronic bronchitis, chronic inflammatory diseases of the upper airways, and chronic colitis. Complex study of the peripheral component of the erythron showed that disorders in erythrocyte morphology and function (decreased erythrocyte dry weight, low concentration of sulfhydryl groups and lipoproteins, decreased content of high molecular weight polypeptides in parallel with increased content of low molecular weight proteins, increased number of transformed cells, and enhanced reversible aggregation) were nonspecific and did not depend on the location of the inflammatory process.

Abscisic acid accumulation and osmotic adjustment in cassava under water deficit

Three cassava ( Manihot esculenta) genotypes were grown in greenhouse conditions and subjected to water deficit treatments to: (a) assess the extent to which abscisic acid (ABA) and osmotically active solutes accumulate in cassava leaves during water deficit and solute contributions to osmotic adjustment (OA), and (b) determine whether cassava leaves substantially increase their content of dehydrin-like polypeptides during water deficit. The concentration of ABA, sugars (sucrose, glucose, and fructose), proline, and potassium salts (K-salts) were determined in mature and expanding leaves. ABA content increased under water deficit, consistent with a putative role in regulating solute accumulation, and thus OA. The extent of osmotic adjustment, although modest, was higher in young folded leaves than in mature leaves. K-salts were the major contributors to total osmolyte concentration in both mature and expanding leaves, accounting for approximately 60% of osmotic potential. The concentration of K-salts increased in response to water stress and was positively correlated with the extent of OA. In contrast, total sugars (sucrose + glucose + fructose) decreased during water deficit, showing a negative correlation with OA. Although the concentration of proline in mature leaves increased in response to water stress its contribution to the total change in osmotic potential was insignificant. Thus, in the current study, cassava used K-salts as its primary osmolyte, and did not substantially adjust its osmotic solute concentration during the 6-day water deficit. Expression of dehydrins was not found in any of the genotypes or leaf stages.

The Delta-Sleep Inducing Peptide in Cerebral Mechanisms of Emotional Stress

The problem of the anti-stressor effect of the delta-sleep inducing peptide (DSIP) and of its neurophysiological mechanisms of action is considered. Physiological data are exposed that indicate an intraventricular administration of DSIP in rats to results in an increase of resistance to emotional stress, according to behavioral and autonomous reactions, and in a decrease of excitability of the brain structures responsible for protective reaction. Radioimmunological evidence is presented for the resistance of animals to emotional stress to depend on the content in hypothalamus of certain oligopeptides, such as β-endorphin, DSIP, and the substance P. It was shown that animals resistant to emotional stress had a higher content of these oligopeptides in hypothalamus than those predisposed to stress. The issue is discussed that the prolonged anti-stressor effects after the DSIP administration are determined by considerable changes of the content of other polypeptides and hormones in hypothalamus and blood, which are involved in the reaction under the effect of DSIP.

Positional effect on protein and oil content and composition of soybeans.

Soybean (Glycine max [L.] Merr.) protein and oil qualities, with respect to monogastric nutrition, have been linked to the relative abundance of specific protein subunits and fatty acids, respectively. An analysis of field-grown soybean seeds by near-infrared spectroscopy revealed significant differences in their protein and oil contents as a function of nodal position. Seed proteins from the plant apex were high in protein and low in oil content, while those from the basal region exhibited an opposite pattern of accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total seed proteins revealed that the beta-subunit of beta-conglycinin content was 4-fold higher in seeds from the apical nodes than in seeds from basal nodes. The glycinin A3 polypeptide content gradually increased in successively lower nodes from the top of the plant. Its accumulation was drastically reduced when nitrogen was applied at specific growth stages. Exogenous nitrogen did not alter the pattern of beta-subunit accumulation, but accrual of the acidic and basic polypeptides of glycinin was diminished. The remaining seed storage protein components were not influenced by nodal position or nitrogen application. Gas chromatographic analysis of fatty acids indicated that only oleic (18:0) and linoleic (18:2) acids showed variability in accumulation at different nodes. Neither the abundance nor the distribution of the fatty acids was altered by nitrogen application.

Autocrine/paracrine action of pituitary vasoactive intestinal peptide on lactotroph hyperplasia induced by estrogen.

Vasoactive intestinal polypeptide (VIP) content is increased in the hyperplastic pituitaries of estrogen (E)-treated rats, thus suggesting that this neuropeptide could mediate the E effect on lactotrophs. E also decreases pituitary TGF-beta1 content, an autocrine/paracrine inhibitor of lactotroph proliferation, and induces pituitary angiogenesis. To elucidate the role of VIP in this context, lactotroph hyperplasia was induced in female Fisher 344 rats by implanting sc pellets of diethylstilbestrol (DES). Twenty-five days later, the rats were treated with three different increasing doses of a VIP receptor antagonist or the vehicle for 5 d. DES treatment resulted in a marked increase of serum prolactin (PRL), pituitary PRL content, PRL mRNA expression, pituitary weight, and pituitary proliferating cell nuclear antigen. DES treatment also increased pituitary VIP content and VIP mRNA levels, but not in the hypothalamus and cerebral cortex. Simultaneously, DES treatment decreased the pituitary TGF-beta1 content and increased the pituitary content of vascular endothelial growth factor. VIP receptor antagonist partially reverted the effect of DES on serum PRL and pituitary PRL, proliferating cell nuclear antigen, TGF-beta1, and vascular endothelial growth factor contents, as well as on pituitary weight, in a dose-dependent relation. These data suggest that pituitary VIP mediates the effect of E on lactotroph hyperplasia, pituitary TGF-beta1, and angiogenesis.

Composition and Characteristic Differences in Photosynthetic Membranes of Two Ecotypes of Reed (Phragmites communis L.) from Different Habitats

As compared with the swamp reed (SR) ecotype of Phragmites communis growing in the desert region of northwest China, plants of the dune reed (DR) ecotype from the same region possessed lower chlorophyll (Chl) content in leaves, and less thylakoids and grana stacks in chloroplasts. Tube gel electrophoresis without stain showed that the contents of Chl-protein (Pro) components related to photosystem 2 (PS2) were markedly lower in the DR thylakoid membranes than in the SR thylakoid membranes, while the contents of Chl-Pro components associated with PS1 were almost the same in both types. SDS-PAGE analysis indicated that the content of polypeptides of the light-harvesting Chl a/b complex of PS2 (LHC2) was lower in the DR thylakoids. Besides, the conformation of LHC2 within the DR thylakoid membranes was also altered as indicated by circular dichroism spectra. Hence in the DR, reduced energy harvesting by declining the size of LHC2 might be responsible for the down-regulated PS2 activity. Chl fluorescence parameters. Fv/Fm and quantum efficiency of PS2 (ΦPS2), were lower in the DR leaves than in the SR ones. However, non-photochemical quenching coefficient (qN) was greater in DR than that in SR, implying other energy dissipation way exists in the DR photosynthetic membranes.

Effects of Prolonged UV-B Enhanced Fluorescent Radiation on Some Marine Microalgae

The eukaryotic unicellular microalgae Chlorella salina, Dicrateria inornata, and Isochrysis galbana were grown under control (fluorescent 20 W m-2) and UV-B enhanced (UV-BE, 0.5 W m-2) fluorescent radiation. The growth rate showed marginal increase under UV-BE. Decrease in protein content was observed in Dicrateria cells but in Chlorella an initial increase up to 4 d and in Isochrysis an increase at days 4 and 5 was noted. The chlorophyll a content showed marked increase in Chlorella and Isochrysis but in Dicrateria a decline was found. UV-BE reduced the photosynthetic activity in all three species, but the reduction was larger in Chlorella and Dicrateria. Fluorescence excitation spectra for F682 in Chlorella cells grown for 5 d under UV-BE showed reduction in all peaks. In contrast to this, in Dicrateria and Isochrysis cells, the 530 and 590 nm excitation peaks increased with an appreciable decrease in the 466 nm peak. SDS-PAGE analysis revealed significant decrease in the contents of 47, 33, and 23 kDa polypeptides in Chlorella cells. In Dicrateria cells, significant loss in the content of 55, 38, and 18 kDa polypeptides was observed. The content of low molecular mass polypeptides (15 kDa) remained unaffected. Isochrysis cells were more stable in preserving the content of thylakoid polypeptides.

Accumulation of Microsomal Polypeptides in Barley Roots during Aluminium Stress

Accumulation of two peripheral membrane polypeptides (20 and 28 kDa) in roots of Al-sensitive (cv. Alfor) and Al-resistant (cv. Bavaria) barley cultivars were analysed during Al stress. Both cultivars were subjected to Al concentration ranging from 0 to 150 µM for 24, 48, 72 and 96 h. Accumulation of both polypeptides was determined 24 h after exposure of plants to Al and content of both polypeptides showed only small depedence upon Al concentration and duration of Al treatment. Although, based on root growth test, Bavaria showed significantly greater resistance to Al than Alfor, analysis of 20 and 28 kDa polypeptide pattern has not revealed significant difference between the two cultivars. However, accumulation of 20 and 28 kDa polypeptides in Alfor was selectively induced by Al treatment because different pH of the root media (pH 3.5 to 6.5) or application of other metals (Cu, Co, or Cd) failed to induce these two bands. On the other hand, accumulation of these polypeptides in Bavaria was induced not only by Al, but also by Cd and in a lesser extent by Co treatment.

Theoretical studies of time-resolved spectroscopy of protein folding.

Recently, we have made significant improvements in the accuracy of calculations of the circular dichroism of proteins from first principles. The quality of these calculations (especially at 220 nm, a key wavelength, where the intensity of the band correlates well with the helical content of polypeptides) has given us confidence to use such calculations to analyse nanosecond molecular dynamics simulations of the folding of polypeptides. We use this combined approach to explore the influence of dynamics on the circular dichroism spectroscopy of polypeptides. We apply it to equilibrium molecular dynamics simulations of two beta-sheet proteins with similar structures, but differing circular dichroism spectra. We analyse a molecular dynamics simulation of the acid-unfolding of myoglobin. For both alpha-helical and beta-sheet conformations, we find that changes in dihedral angles of 30 degrees can change intensities of bands in circular dichroism spectra by up to 5000 degree cm2 dmol(-1). Thus, in isolation, moderate differences in circular dichroism spectra cannot be interpreted uniquely in terms of conformational changes. Examination of individual structures allows us to dissect the influence of conformation on the calculated circular dichroism spectra. Our results are aimed at providing a deeper understanding of the optical properties of proteins. An atomic level connection between molecular dynamics simulations and optical spectroscopy is increasingly desirable as theoretical and experimental studies begin to probe protein folding events reliably on the nanosecond timescale.

Transcript profiling of Escherichia coli using high-density DNA microarrays

The chapter describes the techniques that use for comprehensive transcript profiling of Escherichia coli ( E. coli ) using high-density DNA microarrays. High-density DNA microarrays represent a powerful tool with which to simultaneously measure the expression of every gene in a cell. By identifying the genes that are expressed differentially in response to either specific mutations or modified environmental conditions, we are provided with a view of global responsiveness that improves our understanding of microbial physiology and homeostasis on a genomic scale. This technology complements other methods that measure the cellular content of polypeptides and small molecules. The Escherichia coli genome is composed of more than 4.5 megabase pairs of DNA and is predicted to encode 4290 proteins. Even though this organism has been studied extensively for over 50 years and its genome is completely sequenced, the function of about 40% of these genes remains unknown. High-density gene arrays are now providing important insights into gene function and microbial physiology and are fast becoming an essential tool to link information from genomics, bioinformatics, proteomics, metabolite analyses, and flux analyses into a cohesive “snapshot” of cellular metabolism.

Cerebrospinal Fluid Cytokeratins for Diagnosis of Patients with Central Nervous System Metastases from Breast Cancer

In cases of breast cancer, the diagnoses of parenchymal brain metastases and leptomeningeal carcinomatosis are often delayed (1). New inexpensive biochemical diagnostic initiatives have suggested measurement of cytokeratin tumor markers in the cerebrospinal fluid (CSF) (2)(3). Each type of epithelial cell can be characterized by its cytokeratin polypeptide content because the expression pattern varies with the type of epithelium (4)(5)(6). Malignant epithelial tumors and their metastases maintain the cytokeratin expression typical for the tissue of origin (4)(7)(8). Cytokeratins 8, 18, and 19 are abundantly expressed in adenocarcinoma of the breast, only weakly expressed in epithelial cells of the choroid plexus, and not expressed in nerve, glial, ependymal, and meningeal cells (9). Because neither healthy nor malignant epithelial cells are known to secrete cytokeratins, how they appear in the CSF remains unexplained. Some authors have suggested that cytokeratins are released from the large fraction of dead and dying cells in growing tumors (5), whereas others have suggested that cytokeratins are markers of cell proliferation released during mitosis (10). In either case, release may indicate cell turnover in central nervous system (CNS) metastases from adenocarcinoma. Previous reports have addressed the diagnostic performance of the tissue polypeptide antigen (TPA) assay system in the CSF (2). The system uses polyclonal antibodies, and data indicate that they recognize the rod domain of the cytokeratin 8, 18, and 19 fragments; however, the exact structure of the binding epitope is unknown. Recently, another cytokeratin-based assay system that measures tissue polypeptide-specific antigen (TPS) has become commercially available. The TPS …

Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles

U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B" SNF protein, are highly conserved in metazoans.

Changes in Rubisco and Rubisco activase gene expression and polypeptide content in Pinus halepensis M. subjected to ozone and drought

ABSTRACTThe regulation of ribulose‐1,5‐biphosphate carboxlase/oxygenase (Rubisco) and Rubisco activase was followed for 3 months in an experiment studying the effects of ozone and water stress on Aleppo pine. Rubisco activity was shown to be reduced by 30% in the presence of ozone, whereas no significant effect of water stress was noticed. The effect of combined stresses on Rubisco activity was similar to the effect of ozone. The changes in protein quantity of Rubisco large subunit (LSU) and Rubisco activase (RCA), compared with control plants, were similar to that of the Rubisco activity. Using homologous probes obtained by reverse transcription (RT)‐polymerase chain reaction (PCR), rbcL and rca transcript quantities were quantified during the course of the experiment. RbcL and rca mRNA quantities decreased in ozone and after drought. Changes in rbcL transcript quantity in needles subjected to the combination of ozone and drought were similar to the ones detected when drought was applied alone. On the contrary, the pattern of rca changes under the combination of the two stresses was similar to that of ozone applied alone. A positive correlation existed between the effects of ozone on Rubisco activase and Rubisco LSU protein quantities, which was not so obvious by comparing transcript quantities. This could suggest a potential post‐transcriptional coordinated regulation of the two proteins under stress‐imposed conditions.

Effects of Cyclosporin A on Cell Fusion in a Monkey Kidney Cell Line Persistently Infected with Measles Virus

The authors studied the effects of immunosuppressive peptide cyclosporin A (CsA) on cell fusion efficiency in cells persistently infected with measles virus (448-PI-Vero cells). Treatment of 448-PI-Vero cells with 5 µM CsA enhanced the infusion. In addition, the expression of measles virus antigen on cell surface was increased by treatment with CsA. The addition of phenothiazine, an anti-calmodulin drug, enhanced the fusion of 448-PI-Vero cells in the presence of CsA, although treatment with phenothiazine alone did not affect polykaryocyte formation. The enhancement of fusion efficiency in 448-PI-Vero cells by CsA was suppressed by oligopeptide Z-D-Phe-Phe-Gly, a synthetic oligopeptide that inhibits fusion induced by measles virus. Since the cell content of major virus-specific polypeptides, such as hemagglutinin, nucleoprotein or matrix protein is the same as in untreated controls, this fusion enhancement may be related to transport and accumulation of measles virus glycoproteins.

Diabetes Mellitus is Associated with a Decrease in Vasoactive Intestinal Polypeptide Content of Gastrointestinal Tract of Rat

Vasoactive intestinal polypeptide (VIP) is an inhibitory non-adrenergic, non-cholinergic transmitter, which mediate in the relaxation of sphincters of the gastrointestinal tract. The aim of this study was to determine whether there is a change in the pattern of innervation and tissue content of VIP in the rat gastroduodenum after the onset of streptozotocin (STZ)-induced diabetes mellitus. Diabetes was induced by a single intraperitoneal injection of STZ (60 mg Kg−1). Four weeks after the induction of diabetes mellitus, the rats were anaethetised and the pancreata were removed for further processing. VIP was localized and measured in normal and diabetic rat gastroduodenal tissues by immunohistochemistry and radioimmunoassay, respectively. VIP immunoreactivity was stronger in the ganglion cells of the submucosal and myenteric plexuses of the gastric antrum and duodenum of normal rats (n = 6) when compared to that of diabetic rats (n = 6). Moreover, the number of VIP-positive neurons was significantly lower in the gastrointestinal tract of diabetic rats compared to normal. The VIP content of the gastric antrum and duodenum of diabetic rat was significantly lower (p < 0.05) than that of normal rat. In contrast to the lower tissue levels of VIP in the gastroduodenal segment of diabetic rats, the plasma level of VIP was significantly higher (p < 0.04) in diabetic rat compared to normal. The plasma level of VIP in normal rats was comparable to that measured in normal human beings. A low tissue level of VIP in the gastroduodenal tract of diabetic rat may contribute in part to the abnormal gut motility observed in diabetic patients.