Functional aspects of blood plasma proteins.

The viscosity of solutions (6% w/w protein in 45% sucrose) of porcine and bovine whole blood plasma, porcine serum and porcine plasma fractions exhibited Newtonian behaviour between 20°C and 73°C. On further heating, the viscosity of these solutions increased exponentially producing a reversible gel structure at 76°C and an irreversible gel at 79°C. In contrast, egg albumen protein solutions indicated a relatively low initial viscosity but started to thicken at 65°C, forming reversible gels at 73°C and irreversible gels at 76°C. The viscosity of the egg albumen protein solutions was similar to that of the serum proteins and plasma protein fractions and less than that of whole plasma protein solutions. Viscosities lower than expected were exhibited by mixtures of whole porcine or bovine plasma and egg albumen proteins. However, synergistic interaction between the proteins of egg albumen and porcine plasma fraction I and fraction III (albumin) and porcine serum occurred at high temperatures of heating.     

Effects of Enzyme Choice and Fractionation of Commercial Enzyme Preparations on Protein Recovery in Curd

Five different commercial milk clotting preparations (bovine rennet, calf rennet, calf rennet-porcine pepsin mixture, Mucor miebei protease, and modified Mucor miehei protease) were adjusted to equivalent milk clotting activities and then used to clot milk. Percentages of protein in the resulting wheys were compared. Calf rennet, bovine rennet, or modified Mucor miehei protease caused less loss of protein to the whey than Mucor miehei protease or calf rennet-porcine pepsin mixture. The five enzyme preparations were then fractionated by gel filtration. Fractions with milk clotting activity were pooled. Original enzyme preparations and the pooled fractions made from them were standardized to the same clotting activity, then used to coagulated milk to compare their effect on protein loss to the whey. Fractionation significantly improved protein recovery when bovine rennet and calf rennet-porcine pepsin mixture were used as coagulants but not when calf rennet, Mucor miehei protease, or modified Mucor miehei protease were used.     

Effect of feeding single-dam or pooled colostrum on maternally derived immunity in dairy calves

The role of colostrum management in providing adequate immunological protection to neonatal calves has been widely investigated, and thresholds for colostrum quality, as well as optimum volume and timing for colostrum feeding have been established. However, limited information is available on the effect of colostrum source (single dam or pooled) on passive immunity, as well as subsequent antibody survival in the calf. This study aimed to assess the effect of feeding single-dam colostrum (own and other dam) or pooled colostrum on transfer of passive immunity, and also investigate the rate of depletion of disease-specific antibodies among dairy calves. In total, 320 cows and 119 dairy heifer calves were enrolled in the study. Calves were blood-sampled immediately after birth and received either own-dam, other-dam, or pooled colostrum. Calves were blood-sampled at 24 h to assess serum IgG concentrations and at monthly intervals thereafter to document disease-specific antibody survival. Mean colostrum IgG concentration was higher for other-dam treatment group, whereas own-dam and pooled treatments were similar. For all treatment groups, the mean IgG concentration was >80 mg/mL, exceeding the quality threshold of 50 mg/mL. Mean calf serum IgG concentration was lower for calves fed pooled colostrum compared with those that received colostrum from a single cow. There was a negative association with 24-h serum IgG and calf birth bodyweight; calves <30 kg at birth had the highest 24-h serum IgG concentration. Survival of antibodies to bovine viral diarrhea, Salmonella infection, leptospirosis, bovine parainfluenza 3 virus, bovine respiratory syncytical virus, rotavirus, and coronavirus was not associated with colostrum source; however, antibodies to infectious bovine rhinotracheitis had a greater period of survival among calves fed own-dam colostrum. We found that feeding single-dam colostrum can thus improve calf immunity through increased serum IgG levels and antibody survival rates. Furthermore, we hypothesize that immune exclusion may occur with pooled colostrum; therefore, providing pooled colostrum may still be a good practice as long as it can be ensured that enough antibodies are absorbed into the blood stream to deal with pathogens calves may encounter because different dams may have antibodies against different strains of viruses and bacteria, yielding cross protection.     

Protein kinase C and its endogenous substrate proteins in bovine mammary gland

Protein kinase C activity was evaluated and its endogenous substrate proteins were explored in bovine mammary gland. Activity was detected in both cytosolic and particulate fractions. However, the activity was more abundant in particulate than in cytosolic fractions. By gradient elution (0 to .5 M NaCl) of the enzyme from a DEAE-cellulose column, cytosolic enzyme was eluted at about .2 M NaCl, and particulate enzyme was eluted at .3 to .5 M NaCl, suggesting that the particulate enzyme was different from the cytosolic enzyme. At least four proteins (52, 43, 41, and 35 kdal) were demonstrated for the substrates of the enzyme in the gland. Two (52 and 43 kdal) of the four proteins were mainly detected in particulate fraction, whereas the 35-kdal protein was in cytosolic fraction and the 41-kdal protein was equally distributed in both fractions.     

2种不同牛乳外泌体提取方法的比较研究

目的比较研究2种不同的牛乳外泌体提取方法。方法分别采用超速离心法和蔗糖密度梯度离心法从牛乳中提取外泌体,利用透射电镜、动态光散射仪、纳米颗粒追踪仪、免疫印迹等技术对牛乳外泌体进行全面的鉴定表征。结果利用上述2种方法提取获得的牛乳外泌体呈现双层膜的杯托状结构,总体粒径分布在30～200 nm之间且外泌体中常见的特异标记蛋白CD9、CD81、TSG101均存在,不存在污染蛋白Calnexin。结论超速离心法与蔗糖密度梯度离心法均可从牛乳中提取获得结构完整的外泌体,与超速离心法相比,通过蔗糖密度梯度离心法可获取杂质较少、粒径分布更集中的牛乳外泌体,但该法耗时长、外泌体浓度较低。     

Aqueous matrix compositions and pH influence feline calicivirus inactivation by high pressure processing

The individual effects of pH (pH 3 to 8), NaCl (0 to 21%), sucrose (0 to 70%), and whey protein (0 to 2%) on pressure resistance of feline calicivirus (FCV) in Dulbecco's modified Eagle medium with 10% fetal bovine serum were determined. At pH 3 through 8, the virus was more resistant to pressure at a pH of < or = 5.2. For FCV samples with sucrose (up to 40%) or NaCl (up to 12%), the amount of FCV inactivated by pressure was inversely proportional to the sucrose or NaCl concentration. For example, a treatment of 250 MPa at 20 degrees C for 5 min reduced the FCV titer by 5.1 log PFU/ml without added sucrose and by 0.9 log PFU/ml with 40% sucrose. Reduced pressure sensitivity with increasing NaCl and sucrose concentrations was not a simple function of water activity. Different PFU reductions were observed for NaCl and sucrose samples with equivalent water activity. When protein at concentrations up to 2% did not provide a protective effect. The combined effect of NaCl and sucrose at 4 and 20 degrees C on pressure resistance of FCV also was examined. When both NaCl and sucrose were added to the FCV stock, they had an additive effect on increasing the pressure resistance of FCV. The individual (6% NaCl or 20% sucrose) and combined (6% NaCl plus 20% sucrose) resistance effects did not abrogate enhanced inactivation for pressure treatments at 4 degrees C compared with those at 20 degrees C. Aqueous matrix compositions, in particular different concentrations of NaCl and sucrose or different pH values, can substantially alter the efficiency of virus inactivation by high pressure processing.     

Milk lipoprotein lipase distribution in the major fractions of bovine milk

Raw, bovine bulk tank milk and milks from selected cows were separated by ultracentrifugation into four major fractions: casein, sloughed membrane material, serum, and milk fat globule membrane. Milk lipoprotein lipase activity was measured by the pH stat method and protein determinations were made by the Lowry procedure for each of the four fractions in order to calculate specific activity (units per milligram of protein). In six farm-cooled bulk milk samples stored less than or equal to 24 h, casein had a significantly higher milk lipoprotein lipase total activity, 35.66 units/ml of milk, than all of the fractions. Serum had the next highest activity with 11.69 units/ml of milk. Fluff and milk fat globule membrane had activities of .80 and .41 units/ml of milk, respectively. The specific activity of the fluff was 3.3 milk lipoprotein lipase units/mg of protein, which was significantly higher than the casein and serum fractions in pooled milk. Milks from five cows in midlactation were assayed individually for milk lipoprotein lipase activity and protein content immediately after milking and after 12, 24, 48 and 72 h of cold (4 degrees C) storage. Fresh warm milk was characterized by the absence of fluff. Casein had the highest mean activity (29.91 units/ml), followed by serum (10.25 units/ml) and milk fat globule membrane (.26 units/ml) in the warm milk from the individual cows. Upon cooling to 4 degrees C, significant increases in enzyme activity in the fluff and milk fat globule membrane fractions were observed at 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fractionation of secalins and hordeins by preparative electrophoresis at acid pH

In this report, the optimization of a preparative electrophoretic method to fractionate secalins and hordeins is described. Separation was performed in preparative 7% polyacrylamide gels of 4 cm length at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis (CE). Fractions of B- and C-hordeins were isolated in a single run from barley ethanol extract. %- and P-secalin fractions were isolated from rye ethanol extract. Resolution of preparative separation was maintained at a protein load of up to 30 mg in each run. Each secalin and hordein fraction showed several components of close mobility when analyzed by CE. Fractions from the preparative separation were pooled in such a way that no components from one pool were present in the others. These pooled fractions could be used as starting material for single polypeptide purification. Preparative electrophoresis at low pH allowed a simple separation of %- and P-secalins and B- and C-hordeins from crude material under non-denaturing conditions.     

Comparison of binding proteins of glucocorticoids in mammary tissue and in blood sera from lactating cows.

Supernatant fractions (700 X g) isolated from homogenates of mammary tissue slices from three lactating Holstein cows (slices previously incubated with [hydrogen-3] cortisol for 1 h at 37 C) were electrophoresed on 7% polyacrylamide gels. The majority of radioactivity was in a protein(s) 2.5 to 3 cm from the origin whereas bovine serum incubated with [hydrogen-3] cortisol showed the majority of radioactivity in a protein 5 to 6 cm from the origin. N,N-diethylaminoethyl cellulose chromatography of 700 X g supernatant fluids from three lactating cows revealed that [hydrogen-3] cortisol was associated with a protein component that eluted with .3 M potassium phosphate, pH 8.0. In contrast, [hydrogen-3] cortisol bound to bovine sera eluted as two protein components with .05 and .1 M potassium phosphate, pH 8.0. Approximate molecular weights determined from gel filtration (four lactating cows) and sucrose density studies (two lactating cows) were estimated to be 2.5 X 10(5) to 3 X 10(6) for the 700 X g mammary receptor of cortisol and 6 X 10)4) to 8 X 10(4) for the primary protein which binds cortisol in serum. We conclude that lactating bovine mammary tissue contains a protein(s) capable of binding tritiated cortisol which is unique from the corticosteroid binding proteins of blood.     

Techniques for preparation of bovine mammary smooth membranes

Most procedures for fractionation of lactating mammary tissue membranes have utilized centrifuge tubes of small volume. We report a method for large-scale fractionation of bovine mammary membranes by a zonal rotor. Because of the large volume of the zonal rotor (1350 ml), greater amounts of starting material can be fractionated to obtain greater yields of desired fractions. We report results of linear sucrose gradients to fractionate mammary tissue homogenates and step gradient adaptations to accelerate membrane separations. Assay of membrane fractions for marker enzyme activities demonstrated that zonal fractionation and derived techniques were applicable for large scale preparation with higher yields of selected bovine mammary membranes. Application of this technique to small volumes is discussed for laboratories without a zonal rotor.     

Effect of heat on the distribution of fluorescently labeled plasminogen and plasminogen activators in bovine skim milk

ABSTRACT:  Differentially fluorescently labeled bovine plasminogen (PG-594) and human tissue- and urokinase-type plasminogen activators (tPA-647 and uPA-546) were added to bovine skim milk to track the effect of heat on the location and concentration of these plasmin system components following acid precipitation or ultracentrifugation. In unheated milk, the majority (71.7% to 89.0%) of the added PG and PAs associated with casein micelles or acid curd, and PG-594 in the serum fraction was partially due to associations with nonsedimentable caseins. Heat treatment (85 °C for 16 s) significantly ( P < 0.05) affected distribution of PG-594, tPA-647, and uPA-546, resulting in reduced concentrations of PG and PAs in the serum fractions and reciprocal increases in their levels in the nonsedimentable casein fractions. Overall, almost all of the added PG and PAs (95.9% to 97.5%) became associated with caseins following heat treatment. This is the 1st study to successfully use fluorescent labeling to quantify effects of heat on the location of plasmin components in skim milk.     

Thermal denaturation and coagulation of whey proteins: effect of sugars

The thermal coagulation of unfractionated whey proteins was inhibited by various sugars. The disaccharides, sucrose and lactose, were most effective, and the amino sugar, glucosamine, least effective in this respect. Ultraviolet absorption and light-scattering measurements on the thermal denaturation and coagulation of both unfractionated and individual whey proteins (alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin) showed that sucrose promotes the denaturation of these proteins but inhibits their subsequent coagulation. These results are interpreted in terms of the effect of sucrose on the hydrophobic interactions between solvent and protein.     

An optical biosensor-based immunoassay for the determination of bovine serum albumin in milk and milk products

An automated biosensor immunoassay, exploiting surface plasmon resonance detection, is described for the quantitation of bovine serum albumin (BSA) in milk products. Antibody selection, assay conditions and potential non-specific binding interferences were defined. Analytical performance includes a working range of 10–1000 ng mL⁻¹, a method detection limit of 0.02 mg g⁻¹ in milk, an instrument intermediate precision relative standard deviation (RSD_iR) of 3.7%, an intermediate precision RSD_iR of 8.9% for whey protein concentrate, and a single flow cell stable for at least 400 cycles. Accuracy was demonstrated by recovery, compliance with a certified reference material and comparison with a high performance liquid chromatographic method. The technique was applied to the estimation of BSA content of bovine milk, colostrum, whey protein fractions and infant formulae. The change in BSA expression during early bovine lactation and across a production season, and the thermal denaturation of BSA were also investigated.     

EFFECT OF DECOLORIZATION AND LACTOSE INCORPORATION ON THE EMULSIFICATION CAPACITY OF SPRAYDRIED BLOOD PROTEIN CONCENTRATES

Blood protein concentrates were prepared from the serum and decolorized hemoglobin fractions of bovine blood. The emulsifying capacity of the concentrates was dependent on protein concentration and was not affected by spray drying. However, when the serum protein fraction was subjected to the decolorization treatment, its emulsifying capacity was reduced. Lactose, when added to the serum proteins prior to spray drying, prevented this reduction.     

Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha

An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.     

Lipolytic Enzyme Activity of Macrophages in Bovine Mammary Gland Secretions

The ability of macrophages isolated from the involuted bovine mammary gland and pooled raw milk to secrete lipolytic enzymes was investigated. Macrophages obtained from the involuted gland and maintained in cell culture secreted lipolytic enzymes into culture medium for up to 120 h. Leukocytes in pooled raw milk were separated using Ficoll discontinuous density gradients. Macro-phages secreted lipolytic enzymes into the gradient while fractions containing polymorphonuclear leukocytes and lymphocytes did not possess lipolytic activity. Enzyme activity of macrophages from pooled raw milk averaged .1% of the total milk lipoprotein lipase activity present in the original milk samples. Fresh raw milk with a macrophage concentration increased to 2.5 × 10⁶ cells/ml contained 11.6% higher milk lipoprotein lipase activity after storage for 48 h at 4°C. These results indicate that macrophages isolated from bovine mammary secretions produce lipolytic enzymes that could influence milk lipoprotein lipase activity in raw milk over storage.     

Elevation of bovine serum C-reactive protein and serum amyloid P component levels by lactation

C-reactive protein (CRP) and serum amyloid P component (SAP), which are known to increase in sera from humans and many other animals with acute inflammation caused by infection, toxic drug administration or injury, were previously purified from bovine serum. These serum levels were determined by enzyme-linked immunosorbent assay (ELISA) using specific antiserum to bovine CRP or SAP which was prepared by immunizing rabbits and goats with each purified protein. Among 68 healthy Holstein cows, 45 non-lactating cows had levels of CRP and SAP of 20.6 +/- 1.4 and 27.6 +/- 1.3 micrograms/ml respectively; 23 lactating cows had higher levels of CRP and SAP (76.0 +/- 13.6 and 38.3 +/- 5.5 micrograms/ml respectively). In the latter group, there was a high correlation between milk yield and serum CRP levels (P less than 0.001). From these observations, it was assumed that lactation might stimulate CRP synthesis rather than SAP synthesis in bovine liver as an acute phase reaction, and that CRP might be called a lactation-associated protein.     

Impact of irradiation and immunoglobulin G concentration on absorption of protein and immunoglobulin G in calves fed colostrum replacer

The objective of this study was first to evaluate whether irradiation treatment of a commercial colostrum replacer (CR) affected acquisition of passive immunity. If the irradiation treatment negatively affected the acquisition of passive immunity, the second objective was to evaluate whether an increased total IgG mass, in a single feeding of CR derived from bovine serum fractions, could compensate for this effect. Acquisition of passive immunity was assessed by 24-h serum IgG levels, serum protein levels, apparent efficiency of absorption (AEA) of IgG, and the ability to prevent failure of passive transfer (FPT) in day-old dairy calves fed a single feeding of CR. Single-dose packs of CR were sent to a commercial irradiation facility for electron-beam irradiation at 3 to 7 kGy (low irradiation) or 15 to 20 kGy (high irradiation). Fifty-six Holstein, Jersey, or crossbred calves were randomly assigned to 1 of 5 treatments: 1) 130 g of IgG (460 g of CR), no irradiation; 2) 130 g of IgG (460 g of CR), low irradiation; 3) 160 g of IgG (518 g of CR), low irradiation; 4) 190 g of IgG (575.4 g of CR), low irradiation; and 5) 130 g of IgG (460 g of CR), high irradiation. All CR were reconstituted in water and mixed in a household blender to a constant solids concentration of 18.7%. Increasing doses of irradiation (130 g of Ig with no, low, or high irradiation) resulted in a linear decrease in 24-h serum IgG and AEA of IgG, and increased the percentage of calves with FPT. Increasing the IgG mass in the CR (130, 160, and 190 g of Ig with low irradiation) resulted in a linear increase in 24-h serum IgG and serum total protein levels, and a linear decrease in AEA of IgG. There was no effect of increasing the mass of IgG fed on the percentage of calves with FPT. The correlation between serum IgG and serum total protein at 24 h was positive; however, at 24 h the irradiation treatments reduced the serum IgG-to-serum total protein ratio. In this study, CR isolated from bovine serum, providing 130 g of IgG in the first feeding and receiving either no irradiation or a low irradiation treatment, was sufficient to prevent FPT in calves.     

Mak16p is required for the maturation of 25S and 5.8S rRNAs in the yeast Saccharomyces cerevisiae

The nucleolar Mak16p protein of Saccharomyces cerevisiae has been implicated in 60S ribosome biogenesis. To learn more about the role of Mak16p in this process, ribosomal RNA processing was examined in a mak16-1 temperature-sensitive yeast strain. Steady-state levels of the 25S and 5.8S mature rRNA species dropped dramatically over a 4 h period in the mak16-1 yeast after a shift to the non-permissive temperature, while 18S and 5S rRNA levels decreased only moderately. Ribosomal RNA processing (rRNA) analyses showed that the most prominent defect at the non-permissive temperature was a dramatic decrease in 27SB precursor RNA levels, with no significant increase in the levels of any precursor. These data indicate an essential role for Mak16p in the stability of the 27SB precursor rRNA. Association of Mak16p with the 66S preribosomal complex does not appear to be sufficient for its function, because the mutant Mak16-1p protein was detected in sucrose density gradient fractions corresponding to the 66S pre-RNP complex.     

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.