scholarly journals Effects of intramammary infection on whey proteinograms of sheep during lactation

2015 ◽  
Vol 35 (3) ◽  
pp. 230-236 ◽  
Author(s):  
Vânia F. Lemos ◽  
Eduardo L.S. Guaraná ◽  
José A.B. Afonso ◽  
José J. Fagliari ◽  
Paulo C. Silva ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Biochemical Characterization ◽  
Production Systems ◽  
Polyacrylamide Gel Electrophoresis ◽  
Whey Proteins ◽  
Sodium Dodecyl ◽  
Acid Glycoprotein ◽  
Milk Samples ◽  
Lactation Stage ◽  
Potential Biomarkers

The study aimed to identify potential biomarkers of mammary gland infection in Santa Inês sheep. Commercial flocks of sheep provided the same hygiene, sanitary, and nutritional management under semi-intensive production systems were monitored during the lactation stage-and assessed 15, 30, 60, and 90 days after delivery (through the end of lactation and weaning). The California Mastitis Test (CMT) was performed on the mammary glands. Milk was collected for bacterial examination and protein analysis. Bacterial culture and biochemical characterization of the samples were performed. Forty-two milk samples from healthy glands (negative CMT and bacterial testing) and 43 milk samples from infected glands (positive CMT and bacterial testing) taken at the predefined time points were assessed. A rennin solution was used to obtain the whey. The proteins analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allowed for the quantification of nine whey proteins produced in healthy glands: serum albumin, lactoferrin, IgA, IgG heavy-chain (IgG HC), IgG light-chain (IgG LC), total IgG (IgG HC + IgG LC), α-lactalbumin, β-lactoglobulin, protein with MW 15.000 Da, protein with MW 29.000 Da and eleven whey proteins secreted by infected glands, including haptoglobin and α-1-acid glycoprotein. A comparison of whey proteins between healthy and infected glands showed increases (P<0.05) in the secreted and total contents of all proteins, except for IgG LC and α-lactoalbumin. The most significant changes were observed in α-1-acid glycoprotein, lactoferrin and haptoglobin, which showed three-, five-, and seven-fold increases in secretion, respectively. This study showed that haptoglobin, α-1-acid glycoprotein, lactoferrin, albumin, and the IgA and IgG immunoglobulins may serve as potential biomarkers for mammary gland infection in sheep.

scholarly journals Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding

1982 ◽  
Vol 203 (1) ◽  
pp. 45-50 ◽  
Author(s):  
P M Ahmad ◽  
D S Feltman ◽  
F Ahmad
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Specific Activity ◽  
Simple Procedure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Performic Acid ◽  
Mammary Tissue ◽  
Rat Mammary Gland

A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4′-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity.

scholarly journals Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

1976 ◽  
Vol 159 (1) ◽  
pp. 181-184 ◽  
Author(s):  
N Paskin ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
Subunit Size

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.

scholarly journals Biochemical characterization and purification of HILDA, a human lymphokine active on eosinophils and bone marrow cells

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1618-1623 ◽  
Author(s):  
A Godard ◽  
H Gascan ◽  
J Naulet ◽  
MA Peyrat ◽  
Y Jacques ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Biochemical Characterization ◽  
Bone Marrow Cells ◽  
Cell Clone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pure Material ◽  
T Cell Clone ◽  
Sds Page

Abstract We previously described a lymphokine termed HILDA (for human interleukin DA) produced by T-lymphocyte alloreactive clones after antigenic stimulation. This factor sustains the growth of a murine IL3- sensitive cell line (DA2). In addition, HILDA is a potent activator of eosinophils and displays a burst-promoting activity on human bone marrow. In the present study, HILDA was purified to homogeneity from T- cell clone supernatant using successively sequential concentration, concanavalin A (ConA) affinity chromatography with differential elution (alpha-D glucopyranoside and alpha-D mannopyranoside), high-performance liquid chromatography (HPLC) gel filtration and reverse-phase HPLC. The pure material appeared as a 38-kd glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions. Biologic activity could be recovered from SDS- PAGE gel slices corresponding to the 38-kd band. We conclude from the specificity of the DA-2 cell line and biochemical characteristics described that this lymphokine is different from other known factors produced by human T lymphocytes.

scholarly journals Purification and Biochemical Characterization of the VIM-1 Metallo-β-Lactamase

2000 ◽  
Vol 44 (11) ◽  
pp. 3003-3007 ◽  
Author(s):  
Nicola Franceschini ◽  
Berardo Caravelli ◽  
Jean-Denis Docquier ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Metal Removal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Mediterranean Area ◽  
Exchange Chromatography ◽  
Amino Terminal ◽  
Chromatography Step

ABSTRACT VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla VIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/Km ratios (>106M−1 · s−1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

scholarly journals Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors

1979 ◽  
Vol 181 (3) ◽  
pp. 667-676 ◽  
Author(s):  
M Wohllebe ◽  
D J Carmichael
Keyword(s):  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Neutral Salt ◽  
Guanidinium Chloride ◽  
Lysine Residues ◽  
Cross Links ◽  
Lysine Hydroxylation

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183–188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.

scholarly journals Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk

1982 ◽  
Vol 201 (1) ◽  
pp. 71-79 ◽  
Author(s):  
R Dayal ◽  
J Hurlimann ◽  
Y M L Suard ◽  
J P Kraehenbuhl
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Phosphorus Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Whey Proteins ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Precipitation Line ◽  
Specific Antibodies ◽  
Rabbit Milk

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.

scholarly journals Characterization and purification of H1TF2, a novel CCAAT-binding protein that interacts with a histone H1 subtype-specific consensus element.

1989 ◽  
Vol 9 (4) ◽  
pp. 1566-1575 ◽  
Author(s):  
P Gallinari ◽  
F La Bella ◽  
N Heintz
Keyword(s):  
Cell Cycle ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Promoter ◽  
Molecular Size ◽  
Histone H1 ◽  
Nuclear Extracts ◽  
Definition Of

Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.

scholarly journals 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

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.

Transport of 125I-EGF into milk and effect of sialoadenectomy on milk EGF in mice

1984 ◽  
Vol 247 (3) ◽  
pp. E349-E354
Author(s):  
E. W. Gresik ◽  
H. van der Noen ◽  
T. Barka
Keyword(s):  
Phosphotungstic Acid ◽  
No Reduction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Milk Proteins ◽  
Milk Samples ◽  
Epidermal Growth ◽  
High Concentrations ◽  
Quantitative Changes ◽  
Fold Excess

Epidermal growth factor (EGF) (urogastrone) is found in high concentrations in mouse and human milk. The origin of milk EGF is unknown. Milk samples were collected from lactating mice 2-6 h after the intravenous administration of a tracer dose of 125I-labeled EGF. The milk contained relatively high levels of radioactivity of which 35–46% was precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA) and 26% by a specific antimouse EGF antiserum. Part of the radioactivity in milk was eluted from a Bio-Gel P-10 column at the point at which pure standard 125I-EGF was eluted. These data indicate that the mammary gland of the lactating mouse is capable of sequestering and transporting 125I-EGF into milk. Administration of a thousand-fold excess of unlabeled EGF caused no reduction in TCA-PTA-precipitable radioactivity in milk samples of mice given 125I-EGF. When mice were given 10 micrograms of unlabeled EGF and milk was collected 4 h later, compared with controls the EGF level in milk was doubled. Administration of EGF had no effect on lactose and protein concentrations in the milk, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no qualitative or quantitative changes in the major milk proteins. Milk collected from lactating mice that were sialoadenectomized 6 mo earlier contained about 50% less EGF compared with controls. After the administration of 125I-EGF high concentrations of radioactivity were also found in the mammary and submandibular glands and in the stomach. In the latter organs, however, 95–96% of the radioactivity was in the acid-soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Denaturation of selected bioactive whey proteins during pasteurization and their ability to modulate milk immunogenicity

2020 ◽  
pp. 1-4
Author(s):  
Dimuthu Bogahawaththa ◽  
Todor Vasiljevic
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Polyacrylamide Gel Electrophoresis ◽  
Whey Proteins ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Skim Milk ◽  
Peripheral Blood Mononuclear ◽  
High Temperature Short Time

Abstract This research communication relates to the hypothesis that the consumption of raw or unprocessed cow's milk contributes to lowered prevalence of allergies. Thermal pasteurization of bovine milk can result in denaturation of minor whey proteins and loss of their bioactivity. Denaturation of bovine serum albumin (BSA), immunoglobulin G (IgG) and lactoferrin (LF) in skim milk was studied under different temperature (72, 75 or 78°C) and time (0–300 s) combinations. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that denaturation of all 3 proteins occurred at 72°C and progressed with increase in temperature and holding time. About 59% of LF and 12% of IgG denatured under high-temperature short-time (72°C/ 15 s) pasteurization, while BSA was least impacted. To assess modulation of milk immunogenicity, secretion of selected T helper (Th)-type cytokines by human peripheral blood mononuclear cells (PBMCs) was studied in vitro in response to different concentrations of BSA (0.4–1.0 mg/ml) and IgG (0.8–1.6 mg/ml) in unheated skim milk. Addition of IgG at 1.6 mg/ml induced a prominent Th1-skewed cytokine profile that may not trigger a Th2-skewed allergic reaction. BSA did not appear to modulate milk immunogenicity to any significant extent.

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