Albumin-mediated changes in sperm sterol content during capacitation

The role of albumin in mouse sperm capacitation was studied in relation to its activities as a lipid-solubilizing protein and a sterol acceptor. Two bovine serum albumins (BSA) which supported capacitation, Fraction V and fatty acid-free, both contained cholesterol and phospholipid but were without detectable levels of serum high-density lipoprotein (HDL). The lipid content of BSA could be reduced by trichloroacetic acid (TCA) precipitation; however, removal of all detectable lipids required precipitation with ethanolic acetone and diethyl ether extraction. In medium supplemented with Fraction V, fatty acid-free, or TCA-precipitated BSA, mouse sperm were capacitated as evidenced by their ability to fertilize eggs, concomitant with decreases in total cellular sterol and increases in phospholipid content. Delipidated BSA, fractionated on Sephadex G-100 in guanidine HCl also supported capacitation and mediated a 20% decrease in sperm sterol content, while cellular phospholipid levels remained unchanged. When BSA was modified by cholesterol augmentation, fertilization was inhibited in a cholesterol dose-dependent manner. These findings suggest that modulation of sperm lipid levels comprises an event of capacitation and that albumin mediates this process through its activity as a sterol acceptor.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture

We studied the effect of fetal calf serum and serum protein fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine β-globulins reduced this transfer, although to a lesser extent than whole serum. α-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A γ-globulin fraction failed to exhibit any effect on the uptake of [¹⁴C]phosphatidylcholine by the cells.All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles preincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine to parenchymal cells.These observations were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of the liposomal surface membrane as a result of the binding of certain serum proteins. On the other     

Geographic variation in blood plasma protein concentrations of young herring gulls (Larus argentatus) and Caspian terns (Sterna caspia) from the Great Lakes and Lake Winnipeg

Relative and total amounts of plasma protein fractions are affected by infections, inflammation, and nutritional and physiological status, and are therefore important health indicators in free-living animals. Our objectives were: (1) to examine intercolony differences in plasma protein fractions in prefledgling gulls and terns; (2) to investigate relationships between plasma proteins and other physiological measures such as weight loss, growth, and immune function; and (3) to examine potential associations between organochlorine exposure and plasma proteins. During 1992, blood was collected from 3-week-old herring gull (Larus argentatus) chicks from six sites on Lakes Superior, Huron, Michigan, Erie, and Winnipeg and from 3-week-old Caspian tern (Sterna caspia) chicks from five sites on Lakes Huron, Michigan, and Ontario. These sites provided a wide gradient of organochlorine contamination. Plasma proteins were separated by high-resolution agarose gel electrophoresis and stained with Coomassie brilliant blue dye. Six major fractions were quantified: prealbumin, albumin, α-globulins, β₁-globulins, β₂-globulins, and γ-globulins. Total protein, prealbumin, albumin, and γ-globulin concentrations and the albumin/globulin ratio did not differ among sites. Total protein, albumin, and the albumin/globulin ratio were not decreased in birds experiencing food stress or weight loss. Intersite differences were found in α- and β-globulins. In gulls, β₂-globulins were positively associated with polychlorinated biphenyls (PCBs) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ether (DDE). In terns, PCBs were negatively associated with α-globulins and positively associated with β₁-globulins. Additional research is needed to identify individual proteins and elucidate causal relationships between the particular protein concentrations and factors such as contaminants, growth, and condition.     

Metabolism of lysophosphatidylserine, a potentiator of histamine release in rat mast cells

Lysophosphatidylserine (lysoPS) strongly enhances degranulation of rat mast cells induced by concanavalin A (Con A). In the present paper, the metabolism of exogenous lysoPS in intact mast cells was investigated. Incubation of mast cells with 1-stearoyl-sn-glycero-3-phospho-[3-3H]serine resulted in the rapid binding of lysoPS to mast cells and the time-dependent formation of a considerable amount of [3H]phosphatidylserine. No other radiolabeled lipid metabolites were detected. These results suggest that phosphatidylserine (PS) is synthesized through acylation of lysoPS incorporated into mast cells. Most of the lysoPS associated with mast cells was removed by washing with bovine serum albumin, whereas PS newly formed from lysoPS was not. The cells washed with albumin showed no appreciable histamine release upon subsequent addition of Con A. A different set of experiments was performed using lysoPS analogs which were modified at the hydroxyl group at position 2 of glycerol to avoid acylation. 1-Stearoyl-2-O-methyl-glycero-3-phosphoserine showed almost the same potentiating activity as 1-stearoyl-lysoPS, although the former does not have the free hydroxyl moiety at position 2 of the glycerol residue. The enhancing activity of another lysoPS analog, 1-stearyl-propanediol-3-phosphoserine, which lacks the hydroxyl group altogether, was quite similar to that of 1-stearyl-lysoPS. From these results we conclude that the acylation of lysoPS bears no relation to its potentiating activity and that lysoPS acts toward mast cells as lysoPS itself without any conversion to PS. The effect of replacement of an ester bond at position 1 of glycerol in lysoPS with an ether bond, and the phospholipid composition of rat mast cells are also discussed.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp.

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Interfacial properties of hydrophilic surfaces of phospholipid films as determined by the method of contact angles

Hydrophilic films of phospholipids were deposited onto plastic substrates (surface-treated for cell cultures) and shown to adhere sufficiently for measuring their interfacial properties by the method of contact angles. Both by absolute magnitude and by their dependence on temperature, the interfacial properties of these phospholipid films were indistinguishable from those determined for black lipid bilayer membranes with a different method by other authors. According to both their vesicular micromorphology and water permeability, the surface films can be interpreted to consist essentially of multibilayer vesicles with the hydrophilic groups facing outward. Treatment of these films with cell-culture medium containing calf serum results in changes of interfacial properties that are very similar to those effected on virus-transformed 3T3 cells (earlier work). These interfacial effects may be attributed essentially to serum proteins (such as albumin) adsorbing to phospholipid or cellular surfaces. The interfacial properties of nontransformed 3T3 cells are much less affected by serum treatment (earlier work), which correlates closely with their higher serum requirement for proliferation. Comparison of these results with those on the interfacial effects of serum on phospholipid films suggests that at least part of the proliferation-stimulating effect of serum is mediated by changes of interfacial properties of cell membranes upon adsorption of serum proteins such as albumin. Treatment of phospholipid films with concanavalin A, an inhibitor of cell proliferation, does not result in effects on their interfacial properties correlating with those on cellular membranes. This confirms previous suggestions that the latter depends on specific binding of convanavalin A to specific carbohydrates on the cell membrane.     

Interfacial properties of hydrophilic surfaces of phospholipid films as determined by the method of contact angles. Comparison with cell surfaces

Hydrophilic films of phospholipids were deposited onto plastic substrates (surface-treated for cell cultures) and shown to adhere sufficiently for measuring their interfacial properties by the method of contact angles. Both by absolute magnitude and by their dependence on temperature, the interfacial properties of these phospholipid films were indistinguishable from those determined for black lipid bilayer membranes with a different method by other authors. According to both their vesicular micromorphology and water permeability, the surface films can be interpreted to consist essentially of multibilayer vesicles with the hydrophilic groups facing outward. Treatment of these films with cell-culture medium containing calf serum results in changes of interfacial properties that are very similar to those effected on virus-transformed 3T3 cells (earlier work). These interfacial effects may be attributed essentially to serum proteins (such as albumin) adsorbing to phospholipid or cellular surfaces. The interfacial properties of nontransformed 3T3 cells are much less affected by serum treatment (earlier work), which correlates closely with their higher serum requirement for proliferation. Comparison of these results with those on the interfacial effects of serum on phospholipid films suggests that at least part of the proliferation-stimulating effect of serum is mediated by changes of interfacial properties of cell membranes upon adsorption of serum proteins such as albumin. Treatment of phospholipid films with concanavalin A, an inhibitor of cell proliferation, does not result in effects on their interfacial properties correlating with those on cellular membranes. This confirms previous suggestions that the latter depends on specific binding of concanavalin A to specific carbohydrates on the cell membrane.     

Enhanced sterol synthesis in concanavalin A-stimulated lymphocytes: correlation with phospholipid synthesis and DNA synthesis.

Incorporation of (14C)choline and (3H)myo-inositol into the total lipid fraction, incorporation of (14C)acetate into the sterol fraction and incorporation of (3H)thymidine into DNA were studied in human lymphocyte cultures. Concanavalin A induced an increase in the incorporation of these labels with the following features: (a) Phospholipid synthesis was increased promptly. The lag time for the increase in sterol synthesis and DNA synthesis were 5 hours and 27 hours respectively; (b) The increase in phospholipid synthesis and sterol synthesis was proportional to ConA concentration initially. Cells treated with a high concentration of ConA showed very low levels of DNA synthesis; (c) The increase in phospholipid synthesis could be abolished immediately by alpha-Methyl-Mannoside. alpha-Methyl-Mannoside blunted but did not abolish the increase in sterol synthesis. alpha-Methyl-Mannoside enhanced DNA synthesis of those cells which had been treated by a high concentration of ConA; and (d) Selective inhibition of sterol synthesis with 25-hydroxycholesterol did not prevent the increase in phospholipid synthesis, but it blocked the increase in DNA synthesis. Supplement of LDL, HDL or total lipoproteins to lymphocyte cultures was effective in preventing the inhibition of DNA synthesis by 25-hydroxy-cholesterol. These results suggest that in lymphocyte activation by ConA phospholipid synthesis, sterol synthesis and DNA synthesis were sequentially increased. The rate of cellular commitment to mitogenesis was proportional to ConA concentrations. High concentrations of ConA arrested the cell growth at a postcommitment point in the G1 phase. Enhanced phospholipid synthesis was a precommitment event. Enhanced sterol synthesis was a postcommitment event and reflected the requirement of an increased cholesterol supply for the passage of cell growth through G1.     

Serum proteins in young hedgehogs

Four serum protein components are present at birth: α-, β- and γ-globulins and albumin.
The preferential transfer of γ rather than β-globulin, which occurs both prenatally and postnatally in this species, is reflected in the initial amounts of these proteins present at birth and in the relatively rapid increase in the concentration of γ-globulin which occurs postnatally. By five weeks of age seven protein components are discernible in the serum, which is comparable in this respect to adult serum.
The albumin/globulin ratio changes from about 1.5 at birth to about 0.3-0.4 in the active adult.     

Swelling of Phaseolus Mitochondria Induced by the Action of Phospholipase A

Phaseolus vulgaris mitochondria incubated in sucrose swell rapidly upon the addition of phospholipase A. Bovine serum albumin inhibits the swelling. The release of free fatty acids as a result of phospholipase A action on the mitochondria is detected only in the presence of bovine serum albumin, which promotes the hydrolysis of both mitochondrial phospholipids and purified lecithin. Either free fatty acid or lysolecithin is able to initiate an extensive mitochondrial swelling in sucrose. It is suggested that phospholipase A-induced swelling results from the release of lysophosphatides plus free fatty acids and their subsequent detergent action on the membranes rather than phospholipid loss per se.     

Substrate-specific stimulation of protein kinase C by polyvalent anion

The activity of protein kinase C (PKC) toward arginine-rich substrates was greatly stimulated by sulfate and phosphate, but not by monovalent anions. This stimulation did not require phospholipid, calcium, or diacylglycerol, and appeared to mimic the stimulation by phospholipid. Anionic proteins such as bovine serum albumin also promoted PKC activity toward certain substrates that were characterized by either high arginine or high lysine content. The mechanism of both of these stimulations appeared to be related to formation of a substrate-PKC complex which is essential to phosphorylation by PKC. Polyvalent anions bind the cationic substrate and, together with PKC, form an aggregate which allows phosphorylation. Potential physiological relevance of this stimulation is discussed.     

Immunochemical study of the specific antigens of human amniotic fluid]

Four specific antigens (trophoblastic beta 1-globulin, placental lactogen, alpha 1- and alpha 2-globulins of human placenta) were identified using antisera to the native amniotic fluid. Five antigens with the mobility of prealbumins, alpha 1-globulins, alpha 2-globulins and beta 2-globulins which bear no resemblance with the previously studied antigens were identified using antisera to the acid fraction of the amniotic fluid. Both the prealbumins and alpha 2-globulin were found in the blood serum of foetuses of different age and of newborn infants; these proteins were absent from the blood serum of pregnant women and donors. They received the names of embryonic prealbumine 1, embryonic prealbumine 2 and embryonic alpha 2-globulin. The protein with the mobility of alpha 1-globulins was found in the amniotic fluid of foetuses and in the blood serum of pregnant women only and received the name of amniotic alpha 1-globulin. The concentration of the antigens in question was studied in the developing foetuses and in the blood serum of pregnant women at different stages of pregnancy.     

Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium

The seminal plasma in sperm suspensions from boar, bull, rabbit, ram and stallion was replaced with simple defined media as completely as possible by a combination of centrifugation through Ficoll and dilution. After this process, motility declined and the cells showed a tendency to agglutinate and/or stick to glass. Varying the ionic strength of the medium had little effect upon these parameters but sperm motility was preserved better in the presence of serum albumin. When a number of purified proteins and other macromolecules were tested individually in this way for their motility-preserving ability, bovine or human serum albumin was consistently the most effective. Defatting the albumin or altering its nature by mild reduction, oxidation or alkylation had little detectable effect on its motility-preserving ability; the protein did not appear to be acting as a chelator of metal ions, for it could not be replaced by EDTA. The response of the spermatozoa to replacemrnt of seminal plasma varied between species: bull spermatozoa were particularly sensitive and serum albumin had little effect upon their subsequent motility.     

Inhibition of ubiquitin-dependent proteolysis by non-ubiquitinatable proteins

The effect in reticulocyte lysates of proteins with blocked amino groups on the ATP-dependent degradation of casein and serum albumin was studied in order to assess the extent to which proteins with blocked and with free amino groups share common paths of proteolytic degradation. Completely acetylated or succinylated casein and acetylated or succinylated serum albumin (reduced and carboxymethylated), in addition to other amino-modified proteins, inhibited the ATP-dependent proteolysis of both casein and reduced carboxymethylated serum albumin. Inhibition of serum albumin degradation by acetylated serum albumin was competitive, whereas inhibition of casein degradation by acetylated casein was largely competitive with evidence of mixed kinetics. The different amino-blocked proteins studied, which were largely unfolded under assay conditions, were similarly effective as inhibitors on a weight basis, with Ki values in the range 0.2-0.6 mg/ml; there was no correlation between the ability of the blocked proteins to serve as proteolysis substrates and their effectiveness as inhibitors. Studies of the effects of acetylated proteins on the conjugation of ubiquitin to serum albumin and casein demonstrated that the acetylated proteins blocked formation of ubiquitin-albumin conjugates and of selected casein conjugates; the steady state concentration of selected conjugates of endogenous lysate proteins was increased in the presence of amino-blocked proteins. The results suggest that proteins with blocked amino groups, which cannot serve as substrates for ubiquitin conjugation, can compete for binding to those ubiquitin conjugation factors that recognize and ubiquitinate potential substrates of the ubiquitin pathway. The similar inhibitory properties of the different blocked proteins in turn suggest that a common factor in binding to these conjugation factors may be recognition of the polypeptide backbone.     

Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

Ascaris suum: specific antibodies in isolated intestinal loop washings from immunized swine.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.     

Agarose Gel Electrophoretic Fractionation of Serum Proteins in Adult Cattle. II. A Study Of Cows with Different Diseases

In 283 dairy cows suffering from internal disorders the serum proteins were studied by agarose gel electrophoresis supplemented by total protein and albumin determinations. The clinical diagnoses could be grouped according to protein pattern. Group 1 (abomasal displacement and traumatic muscle injury) did not appreciably affect the serum protein concentrations and represented primarily non-inflammatory diseases or diseases of non-infectious origin. Group 2 (leukosis) occupied an exceptional position, with heavy lowering of albumin unaccompanied by a corresponding rise of total globulin. The γ-globulin concentration was significantly lowered. Group 3 (acute traumatic peritonitis) represented an acute inflammatory process with increase chiefly of the α-globulins, while the γ-globulin concentration was normal. Group 4 (chronic traumatic peritonitis, summer mastitis, chronic subclinical mastitis, chronic laminitis and polyarthritis, urinary tract infection, abscess, and sub-acute-chronic pneumonia) comprised diseases chiefly of chronic inflammatory character and with infectious origin. Especially characteristic was the heavy rise of globulins in general and of γ-globulin in particular. In most cases there was a large increase also of the α- and p-globulin fractions.     

Albumin stimulates the release of arachidonic acid from phosphatidylcholine in hamster lungs

¹⁴C-Arachidonic acid injected into the pulmonary circulation of isolated hamster lungs was effectively incorporated into lung lipids. Once retained the radiolabel was relatively stable but the release of radioactivity increased up to 10-fold when bovine serum albumin (1 %) was added to the perfusate. This efflux of radioactivity was not blocked by quinacrine, a phospholipase A₂ inhibitor. In albumin experiments the released ¹⁴C-araehidonate griginated mainly from the phospholipid fraction in which phosphatidylcholine was the main source of the released radioactivity.Pulmonary infusion of albumin had no significant effect on the amount of ¹⁴C-arachidonic acid in the neutral lipid or free fatty acid fractions of perfused lungs. In experiments with albumin about 80 % of the released radioactivity co-chromatographed with unlabelled arachidonic acid whereas in the absence of albumin only about 20 % of the released radioactivity was unmetabolized arachidonic acid. This study indicates that albumin stimulates the release of arachidonic acid from isolated hamster lungs and that the release is increased mainly from the phosphatidyl choline fraction.     

Haematological and serum protein profiles of Mugil cephalus: effect of two different habitats

The aim of this study was to assess the influence of two different habitats, Faro Lake (group A) and Tyrrhenian Sea (group B), on the haematological and serum protein profiles of Mugil cephalus. Our results showed significant differences of white blood cells, total proteins, prealbumin, albumin and α-globulins between groups A and B. These findings suggest that changes in haematological and serum protein profiles are important indices in monitoring the effects of aquatic habitat changes, representing an adaptive physiological response to different habitats of M. cephalus.