Quantitative aspects of mucus glycoprotein biosynthesis in rat gastric mucosa

The synthesis of the polypeptide backbone of mucus glycoproteins in rat stomach was studied. CsCl centrifugation of the homogenate of [3H]serine pulse-chase labelled stomach or mucosal scrapings showed that [3H]serine was mainly incorporated into molecules having a density identical to that of proteins and that only 8-12% was incorporated into macromolecules with the density of mucus glycoproteins. [3H]-Galactose, however, was almost exclusively incorporated into macromolecules with a density identical to that of mucus glycoproteins. Electrophoretic analysis of the CsCl fraction containing the mucus glycoprotein revealed that 78% of the [3H]serine-labelled macromolecules had an electrophoretic behaviour identical to that of mucus glycoproteins. Thus, only a small portion (about 6-10%) of incorporated [3H]serine was present in the backbone of the mucus glycoprotein. Translation in a wheat germ cell-free system of total RNA derived from both whole stomach and superficial mucosal scrapings, using either [35S]methionine or [35S]cysteine as radioactive amino acid, yielded a wide range of proteins. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, one major translation product of whole stomach RNA had an apparent Mr (43000) identical to that of rat pepsinogen. As this polypeptide could not be found amongst the translation products of RNA from scrapings it probably was pepsinogen. The present data provide strong evidence that the backbone polypeptide of mucus glycoproteins only accounts for a small part of the proteins synthesized by mucus-producing cells.

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Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation

Clinical Chemistry ◽

10.1093/clinchem/39.4.635 ◽

1993 ◽

Vol 39 (4) ◽

pp. 635-640 ◽

Cited By ~ 393

Author(s):

J Risteli ◽

I Elomaa ◽

S Niemi ◽

A Novamo ◽

L Risteli

Keyword(s):

Type I Collagen ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bone Collagen ◽

Collagen Molecule ◽

Type I ◽

Serum Samples ◽

Amino Terminal ◽

Wide Range ◽

Carboxy Terminal

Abstract We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

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Influence of Gemini Surfactants on Biochemical Profile and Ultrastructure of Aspergillus brasiliensis

Applied Sciences ◽

10.3390/app9020245 ◽

2019 ◽

Vol 9 (2) ◽

pp. 245 ◽

Cited By ~ 1

Author(s):

Anna Koziróg ◽

Anna Otlewska ◽

Magdalena Gapińska ◽

Sylwia Michlewska

Keyword(s):

Gemini Surfactants ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Control Sample ◽

Extracellular Proteins ◽

Practical Applications ◽

Aspergillus Brasiliensis ◽

Cationic Gemini Surfactants ◽

Wide Range ◽

Cationic Compounds

In this study, we investigated the activities of hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C6), pentamethylene-1,5-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C5), and their two neutral analogues: hexamethylene-1,6-bis-(N-methyl-N-dodecylamine) (A6) and pentamethylene-1,5-bis-(N-methyl-N-dodecylamine) (A5) at concentrations of ½ MIC, MIC, and 2 MIC (minimal inhibitory concentration) against hyphal forms of Aspergillus brasiliensis ATCC 16404. Enzymatic profiles were determined using the API-ZYM system. Extracellular proteins were extracted from the mycelia and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ultrastructure was evaluated using a transmission electron microscope (TEM). Both groups of surfactants caused changes in the enzyme profiles. Larger changes in the number and concentration of enzymes were noted after the action of non-ionic gemini surfactants, which may have been due to the 100× higher concentration of neutral compounds. Larger differences between the protein profiles of the control sample and the biocide samples were observed following the use of cationic compounds. On the basis of TEM analyses, we found that, with increasing concentrations of compound C6, the mycelium cells gradually degraded. After treatment at 2 MIC, only membranous structures, multiform bodies, and dense electron pellets remained. Based on these results, we concluded that cationic gemini surfactants, in comparison with their non-ionic analogues, could have a wide range of practical applications as active compounds.

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Electrophoretic Analysis of Polypeptides of Plasma Membranes from Bovine Pituitary Gland

Canadian Journal of Biochemistry ◽

10.1139/o74-089 ◽

1974 ◽

Vol 52 (7) ◽

pp. 620-630

Author(s):

André Lemay ◽

Fernand Labrie

Keyword(s):

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Broad Band ◽

Sodium Dodecyl ◽

Electrophoretic Pattern ◽

Apparent Molecular Weight ◽

Electrophoretic Analysis ◽

Polyacrylamide Gel ◽

Protein Components ◽

Membrane Components

Purified plasma membranes from bovine hypophyseal tissue have been fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under various conditions of pH and acrylamide concentrations. The best separation of protein components is achieved at a concentration of 7.5% acrylamide and at pH 7.1. Under these conditions, the electrophoretic pattern consistently shows 36 protein bands ranging in molecular weights from 250 000 to 15 000. Only one broad band, having an apparent molecular weight of 150 000, stains for glycoproteins by the period acid – Schiff technique. After electrophoresis on a two-dimensional polyacrylamide gel system using disc gels containing urea and Triton X-100 in the first dimension and SDS in the second dimension, approximately 45 different protein components can be identified. Less than 12% of the membrane proteins are solubilized by washing the membranes with 1 M KCl or NH4Cl. Denaturating agents like urea and lithium 3,4-diiodosalycilate solubilize 55–60% of membrane components. Adenohypophyseal plasma membranes show an eleetrophoretic pattern completely different from that obtained with membranes isolated from the intermediate or posterior pituitary lobes.

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Calreticulin Biosynthesis and Processing in Human Myeloid Cells: Demonstration of Signal Peptide Cleavage and N-Glycosylation

Blood ◽

10.1182/blood.v90.1.372.372_372_381 ◽

1997 ◽

Vol 90 (1) ◽

pp. 372-381

Author(s):

Gerene M. Denning ◽

Kevin G. Leidal ◽

Valerie A. Holst ◽

Shankar S. Iyer ◽

Doran W. Pearson ◽

...

Keyword(s):

Signal Peptide ◽

Human Lymphocytes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Myeloid Cells ◽

Myeloid Cell ◽

Proteolytic Processing ◽

Peptide Cleavage ◽

Wide Range

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

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A Novel pH and Thermo-Tolerant Halophilic Alpha-amylase From Moderate Halophile Nesterenkonia Sp.Strain F: Gene Analysis, Molecular Cloning, Heterologous Expression and Biochemical Characterization

10.21203/rs.3.rs-299039/v1 ◽

2021 ◽

Author(s):

Nastarn Solat ◽

mohammad shafiei

Keyword(s):

Type Species ◽

Biochemical Characterization ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Alpha Amylase ◽

Moderate Halophile ◽

F Gene ◽

Moderately Halophilic Bacterium ◽

Wide Range ◽

High Concentrations

Abstract A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46 % similarities with closely related type species. Also, the genome sequence shared ANI values below 92 % and dDDH values below 52 % with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (<24%) with functionally characterized recombinant halophilic alpha- amylases. The recombinant alpha-amylase was successfully purified from Ni-NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25–75 °C) and pH (4–9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0–4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca2+ ion had no activating effect, but high concentrations of the ion (40 - 200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg2+, Zn2+, Hg2+ and Fe3+. However, it was inhibited only at very high concentrations of these metal ions. Cu2+ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.

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A Comparative Study of Distribution of Protein and Cholesterol in Various Fractions of Human Semen from Infertile and Fertile Subjects

International Journal of Infertility & Fetal Medicine ◽

10.5005/jp-journals-10016-1046 ◽

2012 ◽

Vol 3 (3) ◽

pp. 78-82 ◽

Cited By ~ 1

Author(s):

MS Srinivas ◽

Vickram Sundaram ◽

M Ramesh Pathy ◽

TB Sridharan

Keyword(s):

Molecular Weight ◽

Comparative Study ◽

Seminal Plasma ◽

Semen Analysis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Semen ◽

Exact Function ◽

Wide Range ◽

Significant Difference

ABSTRACT Aim To elucidate the concentration of the protein and cholesterol in different fractions of human semen from different infertile categories and comparing them with the fertile group. Materials and methods The human semen was collected from different infertile categories including oligoasthenospermia, asthenospermia, azoospermia, normospermia, oligospermia and fertile group. Immediately after collection, the semen analysis was done as per WHO standard protocols. After that, the semen was centrifuged to get the different fractions. Four main fractions were obtained, (1) spermatozoa, (2) debris or material that precipitates at 12 K rpm for 10 minutes, (3) prostasomes which was precipitated at 20K rpm for 120 minutes, (4) seminal plasma. The protein concentration was done by Lowry's method and cholesterol was estimated by diagnostic kit. Results Sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS PAGE) was run for all the categories of semen samples for their seminal plasma and the fertility associated protein was identified. A significant difference was found in the concentration of proteins in all subfractions when compared between control and infertile categories. Almost 86% of the protein was recovered from soluble fraction. In case of azoospermia, the protein content was very low when compared with fertile group. Seminal plasma proteins were visualized by silver staining. The molecular weight of the protein bands were ranging from 6.5 to 205 kDa. The band with molecular weight around 55 kDa was found to be missing in case of oligoasthenospermia. This particular protein is said to be fertility associated protein. The content of cholesterol for different subfraction of the human semen samples from infertile and fertile samples was compared. A wide range of cholesterol was recovered from prostasomes, that too purified. Conclusion A thrive study have to be done in all the subfractions of the semen irrespective of the category of samples to know the exact function of the each subfractions in terms of protein and cholesterol distribution. How to cite this article Sundaram V, Srinivas MS, Rao KA, Pathy MR, Sridharan TB. A Comparative Study of Distribution of Protein and Cholesterol in Various Fractions of Human Semen from Infertile and Fertile Subjects. Int J Infertility Fetal Med 2012;3(3):78-82.

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Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

Mediators of Inflammation ◽

10.1080/0962935031000134905 ◽

2003 ◽

Vol 12 (3) ◽

pp. 179-183 ◽

Cited By ~ 4

Author(s):

E. Jablonska ◽

M. Marcinczyk

Keyword(s):

Monoclonal Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Neutrophils ◽

Enzyme Linked Immunosorbent Assay ◽

Cytoplasmic Proteins ◽

Wide Range ◽

Interleukin 15 ◽

Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

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Limited proteolysis of rabbit cardiac procathepsin D in a cell-free system

AJP Cell Physiology ◽

10.1152/ajpcell.1986.250.4.c589 ◽

1986 ◽

Vol 250 (4) ◽

pp. C589-C596 ◽

Cited By ~ 18

Author(s):

A. M. Smarel ◽

S. W. Worobec ◽

A. G. Ferguson ◽

R. S. Decker ◽

M. Lesch

Keyword(s):

Cathepsin B ◽

Cathepsin D ◽

Intracellular Transport ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Limited Proteolysis ◽

Cysteine Proteases ◽

Proteolytic Processing ◽

Reaction Products ◽

Free System

Rabbit cardiac cathepsin D is initially synthesized as an inactive, apparent molecular weight (Mr) 53,000, pI 6.6 precursor (procathepsin D) that is proteolytically processed during intracellular transport to produce the Mr 48,000 isoforms of active cathepsin D found in cardiac lysosomes. To examine potential proteases responsible for intracellular proteolytic processing, biosynthetically labeled procathepsin D was isolated from rabbit ventricular tissue perfused for 30 min with [35S]methionine. Procathepsin D was then incubated in vitro (40 degrees C, 1-240 min) with active cathepsin D, papain, and cathepsin B, either singly or sequentially, and the reaction products analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Incubation of 35S-labeled procathepsin D with active cathepsin D produced a single reaction product (Mr 51,000; pI 6.2). This limited proteolysis occurred at pH 3-5 and was inhibited by pepstatin. Incubation of 35S-labeled procathepsin D with papain or cathepsin B produced a major reaction product (Mr 48,000; pI 6.4) and a minor form (Mr 50,000; pI 6.0). These reactions occurred at pH 4-7 and were inhibited by leupeptin but not pepstatin. Only the Mr 48,000, pI 6.4 products of papain and cathepsin B-mediated proteolysis comigrated with the most basic isoform of active cathepsin D found in cardiac tissue. In addition, the Mr 51,000 intermediate produced by cathepsin D was susceptible to further limited proteolysis by cysteine proteases with resultant production of a Mr 48,000 product. Thus the intracellular proteolytic processing of rabbit cardiac procathepsin D does not result solely from autocatalysis but requires at least one other protease, possibly cathepsin B.

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Calreticulin Biosynthesis and Processing in Human Myeloid Cells: Demonstration of Signal Peptide Cleavage and N-Glycosylation

Blood ◽

10.1182/blood.v90.1.372 ◽

1997 ◽

Vol 90 (1) ◽

pp. 372-381 ◽

Cited By ~ 28

Author(s):

Gerene M. Denning ◽

Kevin G. Leidal ◽

Valerie A. Holst ◽

Shankar S. Iyer ◽

Doran W. Pearson ◽

...

Keyword(s):

Signal Peptide ◽

Human Lymphocytes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Myeloid Cells ◽

Myeloid Cell ◽

Proteolytic Processing ◽

Peptide Cleavage ◽

Wide Range

Abstract Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

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Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

Biochemical Journal ◽

10.1042/bj1330441 ◽

1973 ◽

Vol 133 (3) ◽

pp. 441-455 ◽

Cited By ~ 16

Author(s):

F. Gonzalez-Mujica ◽

A. P. Mathias

Keyword(s):

Nuclear Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Specific Radioactivity ◽

Electrophoretic Analysis ◽

Soluble Proteins ◽

Adolescent And Young Adult ◽

Adult Rats ◽

Chromosomal Proteins ◽

Liver Nuclei

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.

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