Showing papers on "Bovine serum albumin published in 1988"
TL;DR: Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature.
761 citations
TL;DR: It was demonstrated that precoating with bovine serum albumin and non-proteinaceous macromolecules reduced the phagocytosis of microspheres, whilebovine gamma-globulin, human fibronectin, bovines tuftsin, and gelatin precoates enhanced the phagytosis, which was greatly enhanced by the presence of serum.
Abstract: A variety of biodegradable microspheres were prepared from L-lactic acid, DL-lactic acid, or glycolic acid homopolymers and copolymers of different molecular weights and monomer compositions. Phagocytosis of the microspheres by mouse peritoneal macrophages was studied in cell culture system using scanning electron microscopy as well as light microscopy. The diameter of microspheres prepared was less than 2 microns, regardless of the starting polymers. No dependence of the chemical nature of starting polymers was observed on the extent of phagocytosis of the microspheres by macrophages. Precoating the microspheres with water-soluble macromolecules such as proteins had great influence on phagocytosis by macrophages. It was demonstrated that precoating with bovine serum albumin and non-proteinaceous macromolecules reduced the phagocytosis of microspheres, while bovine gamma-globulin, human fibronectin, bovine tuftsin, and gelatin precoating enhanced the phagocytosis. This trend was not influenced by the presence of serum. Only in the case of gelatin precoating, the phagocytosis was greatly enhanced by the presence of serum as compared to precoating with other proteins. Microscopic observation clearly indicated that the phagocytosed microspheres were gradually degraded in the macrophage interior with the incubation time, leading to release of a fluorescent dye encapsulated in the microspheres. The rate of microsphere degradation in cells could be controlled by changing the molecular weight and the monomer composition of the copolymers comprising the microspheres.
261 citations
TL;DR: The peripheral plasma levels of cholecystokinin (CCK) increased significantly during BSA but not during SPI or RS infusions, Thus, CCK levels were not increased by the inhibition of the proteolytic activity by RS in duodenal juice.
Abstract: The objective of this study was to investigate the effects of high and low inhibitor soybean meals on the duodenal enzyme activities and on the possible regulatory role of gastrointestinal hormones in the pancreatic response. After an overnight fast, 11 healthy volunteers received an intraduodenal infusion of saline for 60 min. This was followed by infusion of either of three test meals: extract of raw soybeans (RS), a low inhibitor soy protein isolate (SPI) or bovine serum albumin (BSA), 10 g/h for 60 min. Then saline was again given intraduodenally for 30 min. Gastric juice was collected continuously and duodenal juice and peripheral blood samples were collected every 10 min. Duodenal chymotryptic activity was severely inhibited by RS, whereas SPI and BSA increased the chymotryptic activity. Tryptic activity showed a transient reduction (55%) during RS infusion, whereas BSA and in particular SPI increased the tryptic activity. No change was seen in amylase activity. The lack of total inhibition of tryptic activity has been studied further and is the subject of the accompanying paper. The peripheral plasma levels of cholecystokinin (CCK) increased significantly during BSA but not during SPI or RS infusions. Thus, CCK levels were not increased by the inhibition of the proteolytic activity by RS in duodenal juice.
189 citations
TL;DR: It is shown that a truncated recombinant protein of 23 kDa still has IgG‐binding capacity and also interacts specifically with human serum albumin (HSA), demonstrating that protein G is a bifunctional receptor.
Abstract: Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity chromatography, using IgG or HSA immobilized on Sepharose, showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the protein suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.
164 citations
TL;DR: The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction.
Abstract: A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, alpha 1-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50 degrees C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction.
157 citations
TL;DR: The present work on tumor vascular permeability has led to the following findings, which confirmed that kinin is generated via the kallikrein‐dependent cascade in the ascitic tumor fluid and blocked this kinin‐generating cascade with Kunitz‐type soybean trypsin inhibitor, and the formation of ascites was suppressed.
Abstract: Enhanced vascular permeability in tumor tissue has profound pathological consequences in tumor biology. However, details of the mechanism involved are not clear. The present work on tumor vascular permeability has led to the following three findings. (i) Ascitic tumor fluid contained kinin (about 1-40 ng/ml), which is known to enhance vascular permeability and induce pain in vivo. (ii) Kinin is generated via the kallikrein-dependent cascade in the ascitic tumor fluid. By blocking this kinin-generating cascade with Kunitz-type soybean trypsin inhibitor the formation of ascites was suppressed. (iii) Blocking of kinin-degrading enzymes (kininases I and II) by an appropriate kininase inhibitor (e.g., captopril) may result in increased permeability, leading to accumulation of the ascitic fluid. This phenomenon was verified by an about 1.2-1.5 fold increase in leakage of 51Cr-labeled bovine serum albumin into the ascites when kininase inhibitors had been administered orally 30 min before intravenous administration of the bovine serum albumin.
150 citations
TL;DR: Findings indicate that the 60-kDa glycoprotein is directly involved in a specific interaction of albumin with the cultured microvascular endothelial cells used in these experiments.
Abstract: Confluent monolayers of microvascular endothelial cells, derived from the rat epididymal fat pad and grown in culture, were radioiodinated by using the lactoper-oxidase method. Their radioiodinated surface polypeptides were detected by NaDodSO4/PAGE (followed by autoradiography) and were characterized by both lectin affinity chromatography and protease digestion to identify the proteins involved in albumin binding. All detected polypeptides were sensitive to Pronase digestion, whereas several polypeptides were resistant to trypsin. Pronase treatment of the cell monolayer significantly reduced the specific binding of radioiodinated rat serum albumin, but trypsin digestion did not. Limax flavus, Ricinus communis, and Triticum vulgaris agglutinins competed significantly with radioiodinated rat serum albumin binding, whereas other lectins did not. A single 60-kDa glyco-protein was precipitated in common by these three lectins and was trypsin-resistant and Pronase-sensitive. Rat serum albumin affinity chromatography columns weakly but specifically bound a 60-kDa polypeptide from cell lysates derived from radioiodinated cell monolayers. These findings indicate that the 60-kDa glycoprotein is directly involved in a specific interaction of albumin with the cultured microvascular endothelial cells used in these experiments.
148 citations
TL;DR: It is shown that the simple diffusion theory is inadequate to explain quantitatively the kinetics of protein adsorption, and specific, conformation-dependent, solute-solvent and solutes-interface interactions also seem to influence the kinetic of adsor adaptation of proteins.
138 citations
TL;DR: The susceptibility of a protein to heme-mediated oxidative damage would appear to be determined by factors such as its affinity for heme, the nature of the amino acids in the vicinity of the bound catalyst and the availability of a free coordination site on the iron.
132 citations
TL;DR: It is shown that E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase when these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. bacteria extracts.
128 citations
TL;DR: It is concluded that the facilitation of nonheme Fe absorption by meat is not a general property of all animal protein but is better explained by the action of one or more specific animal tissues.
TL;DR: Analysis of immune complex material in systemic lupus erythematosus (SLE) sera revealed that in SLE sera, autoantibodies that react with the collagen-like part of C1q are detectable, and this may explain the high percentage of positive results for SLESera in the C1Q SPBA.
Abstract: We investigated the connection between the C1q solid-phase binding assay (C1q SPBA) and double-stranded DNA antibodies, and analyzed the immune complex material in systemic lupus erythematosus (SLE) sera. Comparison with a new monoclonal assay for C1q-bearing immune complexes (the 242G3 assay) revealed that the immune complexes in SLE bind specifically to solid-phase C1q, and not to fluid-phase C1q. The C1q solid-phase binding activity sedimented as 7S IgG, was insensitive to DNase treatment, and could be selectively absorbed by C1q-coupled beads and by bovine serum albumin-anti-bovine serum albumin C1q beads, but not by DNA. Thus, antibodies to double-stranded DNA do not interfere in the C1q SPBA. Isolated IgG from SLE serum precipitated the collagen-like portions, and not the globular, Fc-recognizing portions, of C1q. F(ab')2 fragments of IgG from SLE patient serum were able to bind C1q. These data show that in SLE sera, especially in those with low levels of CH50 and C1q, autoantibodies that react with the collagen-like part of C1q are detectable. Since in the C1q SPBA, the C1q molecule is randomly fixed to the solid phase, we can detect not only immune complexes, but also antibodies that react with the collagen part of C1q; this may explain the high percentage of positive results for SLE sera in the C1q SPBA, in contrast to results of other immune complex assays.
TL;DR: Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cells.
TL;DR: Chemiluminescence and benzoic acid hydroxylation were used to detect oxygen-centred free-radical production by H2O2 and 100 microM-Cu2+ and these results may have biological significance, particularly for proteins in lipid environments.
Abstract: 1. Chemiluminescence and benzoic acid hydroxylation were used to detect oxygen-centred free-radical production by 2.5 mM-H2O2 and 100 microM-Cu2+. Free radicals could not be detected by these methods when H2O2 was replaced with 10 mM-t-butyl hydroperoxide (TBH) or 10 mM-cumene hydroperoxide (CH). The inclusion of the thiol compound dithioerythritol (DTET; 100 microM) increased radical production by H2O2 and Cu2+ as judged by both assays. Mannitol scavenged radicals in the chemiluminescence system in a dose-dependent manner. 2. H2O2, TBH and CH, each with Cu2+, gave rise to substantial fragmentation of the protein bovine serum albumin (BSA). This fragmentation could be increased by the inclusion of DTET. Omission of Cu2+ or the addition of the chelator DETAPAC (diethylenetriaminepenta-acetic acid; 1 mM) lead to virtual abolition of fragmentation. Autoxidized lipid in the presence of Cu2+ caused protein fragmentation by reactions of lipid hydroperoxides. 3. Polyacrylamide-gel electrophoresis in the presence of SDS confirmed that production of fragments had occurred. 4. Susceptibility of BSA to enzymic hydrolysis by two different proteinases acting at pH 5 and pH 7.2 was increased after a limited exposure to hydroperoxides in the presence of Cu2+. 5. These results may have biological significance, particularly for proteins in lipid environments (e.g. membrane proteins and lipoproteins).
TL;DR: The reactivities of o-quinones, derived from tyrosinase oxidation of various catechols, with the cysteine residue in bovine serum albumin are compared to show the yield of protein-bound cysteinylcatechols appears to depend on a competition between the intermolecularucleophilic reaction of sulphydryl groups in protein and the intramolecular nucleophilic Reaction of an amino group in the side chain.
TL;DR: It is found that long-chain bases possess potent inhibitory properties which make them useful probes of signal transduction mechanisms, and data indicate an inhibitory effect subsequent to the generation of second messengers and are consistent with protein kinase C as the locus of action.
Patent•
25 Oct 1988TL;DR: In this article, the use of mature human serum albumin residues 1 to n, where n is between 369 and 419 inclusive, are useful as substitutes for albumin in the treatment of burns and shock in humans, the clearances of undesirable compounds, such as bilirubin from human blood, in laboratory growth media and in HSA assays.
Abstract: Polypeptides corresponding to mature human serum albumin residues 1 to n, where n is between 369 and 419 inclusive, are useful as substitutes for albumin in the treatment of burns and shock in humans, the clearances of undesirable compounds, (such as bilirubin) from human blood, in laboratory growth media and in HSA assays. HSA (1-389) is particularly preferred, although not novel per se. The polypeptides may be produced by recombinant DNA techniques, especially in yeast.
TL;DR: Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum.
Abstract: We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells.
TL;DR: In the method reported here, tannins or other phenolics were applied on chromatography paper and reacted with BSA and unbound BSA was washed off and the protein in the tannin-protein complex was measured spectrophotometrically after staining with Ponceau S. dilatata.
TL;DR: Results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.
TL;DR: The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surrounding cumulus cells, cultured in chemically defined medium with bovin serum albumin in the absence of serum.
Abstract: The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, dended of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, bibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations for 8-bromo-cAMP and forskolin significanly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced. By 24 h, most of the control and dbcAMP-treated oocytes were at anaphase I or beyond, while the majority of oocytes treated with 8-bromo-cAMP or forskolin had only progressed to metaphase I. Prolonged cultures for 44 h revealed no significant difference in the proportion of treated oocytes reaching anaphase I. to metaphase II compared with controls. IBMX inhibited GVBD in a dose-dependent manner following 24-h cultrue, and at low concentrations its effects was similar to that observed for high concentration of 8-bromo-cAMP and forskolin. The timing of observation is thus crucial for interpreting the effects of these compounds on bovine oocyte maturation. The results suggest that elevation of intracellular cAMP induces a transitory inhibition of bovine oocyte GVBD. Although the bovine oocyte plasma membrane contains adenylate cyclase, it may also possess an active phosphodiesterase that could prevent dbcAMP and forskolin from raising intracellular levels of cAMP suffeciently to maintain meiotic arres for any considerable time. As 8-bromo-cAMP is only partially hydrolyzed by phosphodiesterase, there may be more of this analogue available to stimulate cAMP-dependent protein kinase, which could account for the more pronounced effect observed with 8-bromo-cAMP compared with dbc-AMP.
TL;DR: The data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.
TL;DR: A technique for determining the amount of thermally denatured, insoluble protein is described, which can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay.
TL;DR: It appears that serum from previously unexposed animals or animals early during infection can kill smooth B. abortus, an appropriate defense mechanism before the organism becomes intracellular.
Abstract: Studies of the serum bactericidal system in bovine brucellosis were undertaken to investigate the role of the humoral immune response in protection of cattle against the facultative intracellular parasite Brucella abortus. Fresh sera from normal control cattle, infected cattle, and cattle immunized with B. abortus cell envelopes were collected before treatment and during the course of immunization or infection. Normal fresh bovine serum or fresh agammaglobulinemic serum from colostrum-deprived calves was effective in killing smooth virulent B. abortus 2308, but rough strains RB51 (a rough mutant of strain 2308) and 45/20 were much more sensitive to serum. The difference in susceptibility to serum was shown to be correlated with differences in lipopolysaccharide chemotype, with the more resistant strain 2308 having O polysaccharide and the more susceptible strains 45/20 and RB51 lacking O side chains. By treatment of fresh serum with MgCl2 and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] killing was shown to occur via the classical pathway of complement activation. When antibody to B. abortus was present, killing of strain RB51 increased but killing of smooth strain 2308 decreased. The earliest antibody response in serum from infected animals did not interfere with killing. When affinity-purified bovine immunoglobulins specific for B. abortus smooth lipopolysaccharide were added to fresh normal bovine serum, immunoglobulin G1 (IgG1) and IgG2 isotypes blocked killing but IgM and IgA isotypes did not. Thus, it appears that serum from previously unexposed animals or animals early during infection can kill smooth B. abortus, an appropriate defense mechanism before the organism becomes intracellular. At later stages of infection, blocking antibodies predominate.
TL;DR: The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl‐3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr.
Abstract: The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated. Cultures were grown for 15–30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors. The effect of factors was tested at different times (4, 10, 22, and 28 hr). At each time, [methyl-3H]thymidine or [5, 6 -3H]uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37°C. The results obtained indicated that the addition of EGF or FCS significantly stimulated [methyl-3H]thymidine incorporation into DNA, reaching the maximum effect after 22 hr. EGF alone significantly stimulated [3H]uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr.
The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4–10 hr). In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed. On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied.
Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures. The addition of EGF to DMEM + BSA significantly increased cell number, while addition of insulin had no effect. The highest values for cell number were observed when insulin and transferrin were added together with EGF. Moreover, EGF treatment in the presence of serum significantly increased the incorporation of labeled amino acids into vimentin, glial fibrillary acidic protein, and actin.
TL;DR: The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.
Abstract: A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.
TL;DR: The results suggest that the destruction of T. cruzi trypomastigote or amastigotes by eosinophils may result from toxic mechanisms involving several granule proteins.
Abstract: The eosinophil granule major basic protein, the eosinophil cationic protein, and the eosinophil-derived neurotoxin were found to be lytic for Trypanosoma cruzi trypomastigotes from blood, cell cultures, or insect vectors and for cultured amastigotes. The toxic effects of the major basic and cationic proteins were inhibited by the polyanions heparin and dextran sulfate, in keeping with the cationic nature of these proteins, or by heat denaturation, suggesting that molecular conformation was also relevant. The lytic activity of the neurotoxin was not inhibited by heating at 56 degrees C for 4 hr, establishing an additional difference with the eosinophil cationic protein. Heparin had only a slight inhibitory effect on the toxicity of the neurotoxin, and dextran sulfate was inactive even at 25 mg/ml. Although both the eosinophil cationic protein and the neurotoxin possess ribonuclease activity, only the toxicity of the latter was abolished by the ribonuclease inhibitor RNasin (Promega, Madison, Wisconsin) or by a competitive substrate, yeast ribonucleic acid. Eosinophil peroxidase significantly increased the extent of trypomastigote or amastigote killing by hydrogen peroxide in the presence of iodide. This effect was abrogated by sodium azide, bovine serum albumin, or gelatin, known inhibitors of the eosinophil peroxidase + halide + hydrogen peroxide system. These results suggest that the destruction of T. cruzi trypomastigotes and amastigotes by eosinophils may result from toxic mechanisms involving several granule proteins.
TL;DR: The results show that alkyl vinyl sulfones have a strong chemical affinity for protein functional groups.
Abstract: Disulfide bonds of bovine serum albumin and wool were reduced by n-tributylphosphine to sulfhydryl groups that were then modified by methyl or ethyl vinyl sulfone in a nucleophilic addition reaction to S-(beta-ethylsulfonylmethyl)-L-cysteine and S(beta-ethylsulfonylethyl)-L-cysteine, respectively. The reductive alkylation was carried out either simultaneously, with both the reducing and alkylating agents present in the reaction mixture, or sequentially, with the reduced proteins first isolated before alkylation. Amino acid analysis studies showed that authentic, synthetic S-(beta-ethylsulfonylethyl)-L-cysteine eluted as a well-resolved peak after serine but that the peak associated with the corresponding methyl derivative overlapped the corresponding peak due to threonine. The extent of alkylation of the sulfhydryl groups of cysteine, epsilon-NH2 of lysine, and NH groups of the imidazole ring of histidine was also measured by amino acid analysis. The results show that alkyl vinyl sulfones have a strong chemical affinity for protein functional groups.
TL;DR: The desialylated bovine caseinoglycopeptide acts as a potent and specific inhibitor of oral Actinomyces adhesion to cell membranes (a soft surface) and could be used as a probe for the study of recognition mechanisms mediated by Actincyces galactose-binding lectins.
Abstract: Various caseinoglycopeptide derivatives prepared from mammalian milk were evaluated as inhibitors of hemagglutinations mediated by Actinomyces viscosus Ny1, Streptococcus sanguis OMZ9, and, for comparative purposes, plant lectins from Arachis hypogaea and Bauhinia purpurea. It was found that recognition of the beta-D-galactose-(1----3)-2-acetamido-2-deoxy-D-galactose carbohydrate chain by Actinomyces viscosus Ny1 organisms and Arachis hypogaea and B. purpurea agglutinins had similar structural requirements; in all cases, the desialylated bovine caseinoglycomacropeptide, on which several units of the above mentioned disaccharide are clustered, behaved as the most potent hemagglutination inhibitor. By contrast, none of the preparations tested inhibited erythrocyte agglutination by S. sanguis OMZ9. Thus, the desialylated bovine caseinoglycomacropeptide acts as a potent and specific inhibitor of oral Actinomyces adhesion to cell membranes (a soft surface) and could be used as a probe for the study of recognition mechanisms mediated by Actinomyces galactose-binding lectins. During the present study, both native and desialylated variants of the same bovine glycomacropeptide also totally prevented the adhesion of Actinomyces viscosus Ny1, S. sanguis OMZ9, and S. mutans OMZ176 to polystyrene surfaces. Comparative evaluations of various structurally different compounds gave the following results. Neither mono- nor disaccharides related to caseinoglycopeptide carbohydrates prevented adhesion; highly positively or negatively charged polypeptides and polysaccharides were either not or only moderately active. Besides these glycomacropeptides, an inhibitory activity was also exhibited by other mucin-type glycoproteins carrying short O-linked carbohydrate chains (including bovine submaxillary mucin), polyethylene glycol, and bovine serum albumin. Consequently, caseinoglycopeptide prevention of oral bacterial adhesion to polystyrene tubes (a hard surface) takes place with no species specificity and can be compared to nonspecific inhibition exhibited by various polymers with very different structural characteristics.
TL;DR: The human hepatic ferritin receptor may play an important role in the uptake of iron into the hepatocyte in physiological and pathological conditions.