Distribution of plasminogen activator in different fractions of bovine milk

1995 ◽  
Vol 62 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Jeffrey H. White ◽  
Boris Zavizion ◽  
Kristen O'Hare ◽  
James Gilmore ◽  
Ming R. Guo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Somatic Cells ◽  
Bovine Milk ◽  
Skim Milk ◽  
Raw Milk ◽  
Sds Page ◽  
Predominant Form ◽  
Cell Pellet ◽  
Molecular Masses ◽  
Milk Casein

SUMMARYThe type and relative amounts of plasminogen activator (PA) in different fractions of bovine milk obtained from 15 Holstein cows were examined. Raw milk was centrifuged to separate skim milk and a somatic cell pellet. PA was mainly localized within the casein fraction, being 42 times that in the serum, and in association with somatic cells. The predominant form of PA in milk casein was isolated from SDS-PAGE gel extracts and had a molecular mass of ∽75 kDa. Its activity was increased 41-fold (P < 0·01) in the presence of fibrin but was unaffected by the presence of amiloride, indicating that it was due to tissue-PA. The predominant forms of PA associated with milk somatic cells were isolated from SDS-PAGE gel extracts and had molecular masses of ∽ 30 and ∽ 50 kDa. The activity of both proteins was unaffected by the presence of fibrin but was dramatically reduced by the presence of amiloride, indicating that they represented urokinase-PA.

Interactions between the bovine milk fat globule membrane and skim milk components on heating whole milk

1992 ◽  
Vol 59 (2) ◽  
pp. 187-195 ◽  
Author(s):  
Avis V. Houlihan ◽  
Philippa A. Goddard ◽  
Stephen M. Nottingham ◽  
Barry J. Kitchen ◽  
Colin J. Masters
Keyword(s):  
Milk Fat ◽  
Bovine Milk ◽  
Skim Milk ◽  
Milk Proteins ◽  
Raw Milk ◽  
Membrane Lipid ◽  
Milk Fat Globule Membrane ◽  
Whole Milk ◽  
Milk Fat Globule ◽  
Fat Globule

SummaryHeating raw milk at 80 °C for 2·5–20 min was found to result in compositional changes in the milk fat globule membrane (MFGM). The yield of protein material increased with the duration of heating, owing to incorporation of skim milk proteins, predominantly β-lactoglobulin, into the membrane. Lipid components of the MFGM were also affected, with losses of triacylglycerols on heating.

The role of various milk fractions and the importance of somatic cells in the formation of germinant(s) for Bacillus cereus when milk is pasteurized

1977 ◽  
Vol 44 (3) ◽  
pp. 555-568 ◽  
Author(s):  
F. L. Davies
Keyword(s):  
Bacillus Cereus ◽  
Cell Count ◽  
High Speed ◽  
Serum Proteins ◽  
Somatic Cells ◽  
Skim Milk ◽  
Raw Milk ◽  
Cell Counts ◽  
Blood Serum Proteins ◽  
Fluff Layer

SummaryConfirmation was obtained for the occurrence of an interaction between high and low molecular weight fractions of milk during its pasteurization, resulting in the formation of germinant(s) for Bacillus cereus. Various milk fractions were added to supernatants or dialysates of raw skim-milk, pasteurized and assayed for germinant. Micellar casein obtained by high speed centrifugation of raw milk stimulated germinant formation, but whole casein obtained by acid precipitation was less stimulatory and individual casein fractions showed no stimulation. A membrane rich ‘fluff’ layer obtained by high speed centrifugation of raw milk was highly stimulatory and suggested the possibility that the somatic cell count of milk may be related to germinant production.Using milk from endotoxin infused and infected quarters of individual cows, a correlation between cell count and germinant formation was clearly demonstrated. Adjustment of the cell counts of individual milk samples was generally accompanied by corresponding changes in germinant formation, though removal of cells by filtration did not have this effect, possibly since germinant precursor had leached from the cells and remained in the milk. Pasteurization was necessary, not simply to express germinant from cells but to effect an interaction of germinant precursors. It is not clear whether the cell associated germinant precursor is derived from the cells themselves or from a related component. Addition of blood serum proteins was without stimulatory effect.Since low cell count milks sometimes supported appreciable germination after pasteurization, but showed still higher levels when the cell count was raised, germinant may be formed via 2 pasteurization dependent processes, one between milk constituents alone and the other involving both milk constituents and somatic cells.

scholarly journals Centrifugation does not remove bacteria from the fat fraction of human milk

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lisa F. Stinson ◽  
Jie Ma ◽  
Alethea Rea ◽  
Michael Dymock ◽  
Donna T. Geddes
Keyword(s):  
Human Milk ◽  
High Speed ◽  
Bovine Milk ◽  
Skim Milk ◽  
Bacterial Dna ◽  
Variable Quantity ◽  
Cell Pellet ◽  
Fat Fraction ◽  
Dna Profiles ◽  
Future Work

AbstractAnalysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Staphylococcus epidermidis was significantly more abundant in the cell pellet compared to the fat fraction (P = 0.038), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.

Levels and location of adenosine 5′-triphosphate in bovine milk

1980 ◽  
Vol 47 (1) ◽  
pp. 91-96 ◽  
Author(s):  
Thomas Richardson ◽  
Thomas C. A. McGann ◽  
Robert D. Kearney
Keyword(s):  
Calcium Phosphate ◽  
Amorphous Calcium Phosphate ◽  
Liquid Scintillation ◽  
Biological Systems ◽  
Scintillation Counter ◽  
Somatic Cells ◽  
Bovine Milk ◽  
Skim Milk ◽  
Milk Samples ◽  
Milk Serum

SummaryBovine milk systems were analysed for adenosine 5′-triphosphate (ATP) using the luciferase-ATP reaction in a liquid scintillation counter. Approximately 0·2 µmol ATP/1 milk serum were evident both in whole milks and the corresponding skim-milks. ATP was not detectable in skim-milk ultrafiltrates. These findings indicated that ATP was present in a non-dialysable portion of skim-milk. Centri-fugation of whole milks from individual cows at 5500 g for 15 min at 10°C yielded skim-milks essentially devoid of somatic cells and bacteria. However, the ATP in the skim-milks decreased by less than 20% compared with the whole milks indicating that the calcium phosphate-citrate (CPC)–caseinate micelles were the source of the ATP. ATP was not detectable in colloidal phosphate-free milk, from which CPC had been removed, confirming that the ATP was sequestered in the constituent CPC. Likewise, the occurrence of significant amounts of Mg, another potent stabilizer of amorphous calcium phosphate (ACP) in other biological systems, was confirmed in the colloidal phosphate of milk. From 0·13 to 0·31 µmole ATP/1 (mean, 0·23) was found in the 9 milk samples studied. The discovery of small but appreciable levels of ATP in the CPC of milk provides further evidence for the analogy previously shown to exist between the CPC complex of milk and the ACP which accumulates in mitochondria. The latter has been postulated to provide an essential precursor for crystalline bone salts to form in ordered calcification processes. The implications of these findings in the biosynthesis of milk are briefly discussed.

scholarly journals PROTEOLYTIC POTENTIAL OF PSEUDOMONAS FLUORESCENS ISOLATED FROM REFRIGERATED RAW MILK

2014 ◽  
Vol 4 (2) ◽  
Author(s):  
Cláudia Lúcia Oliveira Pinto1 ◽  
Solimar Gonçalves Machado ◽  
Rodrigo Rezende Cardoso ◽  
Rita Maria Alves ◽  
Maria Cristina Dantas Vanetti
Keyword(s):  
Pseudomonas Fluorescens ◽  
Proteolytic Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heat Stability ◽  
Raw Milk ◽  
Activity Measurement ◽  
Sds Page ◽  
Milk Casein ◽  
Thermal Stability Of

The growth rate and the proteolytic activity of Pseudomonas fluorescens strains 07A and 041, isolated from cow’s milk, were evaluated at 2, 4, 7 and 10ºC. P. fluorescens promoted protein degradation during storage of milk samples as observed by Proteolytic activity measurement, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and heat stability of milk. Casein hydrolysis resulted in loss of thermal stability of milk and in formation of fragments of low and medium molecular mass. Temperatures up to 10°C did notguarantee raw milk quality when contamination by P. fluorescens was equal or higher than 106 cfu/mL.

Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

1990 ◽  
Vol 63 (01) ◽  
pp. 067-071 ◽  
Author(s):  
Joan C Castellote ◽  
Enric Grau ◽  
Maria A Linde ◽  
Nuria Pujol-Moix ◽  
Miquel LI Rutllant
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Human Peripheral Blood ◽  
Direct Interaction ◽  
Apparent Molecular Weight ◽  
Fibrinolytic System ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Single Chain ◽  
Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

Proteinase and phospholipase activities and development at different temperatures of yeasts isolated from bovine milk

2011 ◽  
Vol 78 (4) ◽  
pp. 385-390 ◽  
Author(s):  
Priscilla A Melville ◽  
Nilson R Benites ◽  
Monica Ruz-Peres ◽  
Eugenio Yokoya
Keyword(s):  
Dairy Products ◽  
Room Temperature ◽  
Bovine Milk ◽  
Raw Milk ◽  
Pathogenic Potential ◽  
Proteinase Production ◽  
Different Temperatures ◽  
Quality Of Milk ◽  
Means Of Transmission

The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

scholarly journals The mechanism of in vitro clot lysis induced by vascular plasminogen activator

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1331-1337 ◽  
Author(s):  
C Tran-Thang ◽  
EK Kruithof ◽  
F Bachmann
Keyword(s):  
Plasminogen Activator ◽  
Whole Blood ◽  
Apparent Molecular Weight ◽  
Clot Lysis ◽  
Initial Concentration ◽  
Low Concentrations ◽  
Sds Page ◽  
Tissue Plasminogen ◽  
Low Rate

Abstract The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v- PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.

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