Baboon serum is superior to human or bovine serum albumin for baboon sperm capacitation and zona binding

Background  Baboon in vitro fertilization requires capacitated sperm in appropriate media. In this study, we compared the effect of baboon serum (Bas), human serum albumin (HSA) and bovine serum albumin (BSA) on baboon sperm capacitation.
Methods  Five males (n = 5) were electroejaculated and 43 oocytes retrieved from super-ovulated female baboons (n = 10). Each sperm sample was assessed for initial motility and concentration before and after swim-up. For swim-up, each sperm sample was incubated separately in Biggers–Whitten–Whittingham media containing either BaS, HSA, BSA or without protein supplementation (control). After swim-up, each sperm aliquot was incubated with two to three oocytes. The number of sperm bound to the zona was evaluated after overnight incubation.
Results  Sperm motility and zona binding was significantly higher after capacitation in media supplemented with BaS than in HSA or BSA or in media without protein supplementation ( P  < 0.05).
Conclusion  Baboon serum is superior to HSA or BSA for baboon sperm capacitation and zona binding.     

Tumour localization of uroporphyrin isomers I and III and their correlation to albumin and serum protein binding

We were the first to report the superiority of uroporphyrin I (UROP I) as a tumour localizer when compared to haematoporphyrin derivative (HPD). In this study, we compared both isomers of UROP, i.e. I and III, in a KHJJ mammary carcinoma mouse model. Six and 18 h after UROP administration, the tumour, skin and gut porphyrin (P) content was quantitated. Tumour UROP I levels were always at least 50% higher than UROP III in tumour, whereas both isomers were barely detectable in the skin and gastrointestinal tract. We then explored the possibility that tumour P uptake might relate in part to the affinity of circulating P to mouse serum proteins (MSP), in particular, the major binding protein constituent, albumin. Copro-P III, deutero-P 2,4 disulphonic acid (DP), proto-P IX (PP) and heptacarboxylic P I (Hepta I) which in our mouse tumour model do not localize in malignant tissue, were compared to UROP I and III. The P was mixed with 0.775 microM human serum albumin (HSA) at different molar ratios (HSA:P range 2-8) and the unbound P concentration quantitated using an Amicon CF-25 membrane cone with centrifugation. The percentage free P was significantly higher for UROP I (92-98%) than III (82-95%) and significantly more than that observed with non-tumour localizing P studied. Similar data were obtained with MSP. This is consistent with the notion that enhanced uptake and retention (particularly UROP I) by malignant neoplastic tissue might reflect a higher affinity for UROP by tumour constituents than by circulating proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.0 nmol/hr/10(6) cells, which is the same as the activity in cord blood lymphocytes (7.0 +/- 2 nmol/hr/10(6) cells). The activity rose twofold to 13.2 +/- 3.8 nmol/hr/10(6) cells at 6 mo of age (p less than 0.001, paired t-test), which is similar to the activity in adult peripheral blood lymphocytes (14.1 +/- 6.3 nmol/hr/10(6) cells). This increased activity in total lymphocytes reflects increased activity in the B cell population. B cell ecto-5'-nucleotidase activity in two infants at 12 to 13 mo of age was 19.3 and 25.2 nmol/hr/10(6) cells, values that are four-to fivefold higher than for cord blood B cells (5.6 +/- 2.8 nmol/hr/10(6) cells) and within the normal range for adult B cells (27.9 +/- 12 nmol/hr/10(6) cells). In spite of a greatly expanded peripheral blood B cell population, studies of immunoglobulin biosynthesis in vitro demonstrated that infant peripheral blood B cells are functionally immature with no synthesis of IgG in response to Epstein Barr virus. Thus, the increase in peripheral blood B lymphocyte ecto-5'-nucleotidase activity in infants precedes their acquisition of a capacity for IgG synthesis in vitro. Data from a hypogammaglobulinemic infant revealed a persistently low ecto-5'-nucleotidase activity over a 10-mo period until at 14 mo of age the activity was 8.8 nmol/hr/10(6) cells in total lymphocytes and 13.0 nmol/hr/10(6) cells in B cells, which correlated with in vivo and in vitro evidence of delayed B cell maturation. Thus, ecto-5'-nucleotidase activity may be a useful cell surface marker in studies of human postnatal B cell maturation.     

Differential regulation of FGF-1 and -2 mitogenic activity is related to their kinetics of binding to heparan sulfate in MDA-MB-231 human breast cancer cells

The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth.     

Effects of tobacco glycoprotein (TGP) on the immune system. III. The effect of aging on the mitogenic response of human peripheral blood lymphocytes to TGP

We have shown that the mitogenic response of human peripheral blood lymphocytes (PBL) to tobacco glycoprotein (TGP), a glycoprotein rich in rutin or rutinlike polyphenol moieties, which is isolated from cured tobacco leaves, does not decrease with age. In contrast, the proliferative response to lipopolysaccharide (LPS) tends to decline with age, and a significant decrease is observed in the mitogenic response to rutin-bovine serum albumin (R-BSA). LPS and R-BSA are similar in some aspects to TGP, the former in that TGP and LPS are both T-independent B-cell mitogens for mice and are both highly negatively charged, and the latter in that covalently bound polyphenol groups are present on both R-BSA and TGP. Although the kinetics of the mitogenic responses to these three mitogens are similar (T. Francus, R. F. Klein, L. Staiano-Coico, G. W. Siskind, and C. G. Becker, Effects of tobacco glycoprotein (TGP) on the immune system. II. TGP is a mitogen for human peripheral blood lymphocytes. Submitted for publication.), multiple regression analyses show no correlation in the mitogenic responses of PBL from young donors to TGP and LPS, to TGP and R-BSA, or to LPS and R-BSA. In contrast, there are significant correlations between the proliferative responses to these mitogens by PBL from old donors. The results suggest that only a small subpopulation of the cells which are stimulated by R-BSA and LPS are not altered with age, and these are most likely the cells that are stimulated by the three mitogens studied.     

An external loop region of domain III of dengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cells

Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.     

beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein.

Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.     

Cytidine 5'-monophospho-N-acetylneuraminic acid or a related compound is the low Mr factor from human red blood cells which induces gonococcal resistance to killing by human serum

A low-Mr factor which induces gonococcal resistance to complement-mediated serum killing has been partially purified from lysates of mixed red and buffy coat cells from human blood. The lysates were dialysed against Tris buffer for 24 h at 25 degrees C with the diffusate being continuously recycled through a column of QAE-Sephadex A25. After elution in an NaCl gradient, the active fractions were both desalted and further purified on Sephadex G10. A second fractionation on QAE-Sephadex A25 and desalting with Sephadex G10 preceded further purification by repeated high-pressure liquid chromatography (HPLC) using a DEAE anion exchange column and desalting with Sephadex G10. Less than 500 micrograms of material showing one peak in HPLC was obtained from 1 litre of blood. After NMR had indicated the possible presence of pyrimidine nucleotide, carbohydrate and N-acetyl groups, nanogram quantities of a commercial preparation of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) were shown to induce gonococci to serum resistance. The synthetic CMP-NANA also co-eluted with the preparation from blood cells in HPLC, and the two materials were indistinguishable in their patterns of acid and heat lability. Furthermore, the resistance-inducing activity of both materials was inhibited by cytidine monophosphate, which is known to inhibit sialylation reactions by CMP-NANA. It appears therefore that the resistance-inducing factor is CMP-NANA or a closely related compound. If the factor is CMP-NANA, biological activities indicated that the cell lysate from 1 litre of blood contained about 40 micrograms, and the most purified preparation contained only about 1%. With this minute amount in a mixture, the presence of CMP-NANA or a closely related analogue could not be established unequivocally by NMR.     

The R28 protein of Streptococcus pyogenes is related to several group B streptococcal surface proteins, confers protective immunity and promotes binding to human epithelial cells

The R28 protein is a surface molecule expressed by some strains of Streptococcus pyogenes (group A streptococcus). Here, we present evidence that R28 may play an important role in virulence. Sequence analysis demonstrated that R28 has an extremely repetitive sequence and can be viewed as a chimera derived from the three surface proteins Rib, alpha and beta of the group B streptococcus (GBS). Thus, the gene encoding R28 may have originated in GBS. The R28 protein promotes adhesion to human epithelial cells, as shown by experiments with an R28-negative mutant and by the demonstration that antibodies to highly purified R28 inhibited adhesion. In a mouse model of lethal intraperitoneal S. pyogenes infection, antibodies to R28 conferred protective immunity. However, the virulence of an R28-negative mutant was similar to that of the parental strain in the intraperitoneal infection model. Together, these data indicate that R28 represents a novel type of adhesin expressed by S. pyogenes and that R28 may also act as a target for protective antibodies at later stages of an infection. We consider the hypothesis that R28 played a pathogenetic role in the well-known epidemics of childbed fever (puerperal fever), which were caused by S. pyogenes. A role for R28 in these epidemics is suggested by epidemiological data.     

Lectin activity of the major surfactant protein (SP-A) may participate in, but is not required for, binding to rat type II cells.

Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.     

Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)

The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hepatocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular trafficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mutation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the constitutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.     

Marmoset red blood cell receptor for membrane-associated complement components is not related to human CR1: partial characterization of the C3-binding proteins responsible for the spontaneous rosette formation between marmoset red blood cells and human leukocytes

Cells from all the human B-lymphoblastoid cell lines tested and most human monocytes form rosettes with marmoset red blood cells (MaRBC). Because previous reports suggested the involvement of complement components in this phenomenon, the mechanism of rosette formation and the eventual similarities between the MaRBC receptor and the CR1 receptor present on human erythrocytes have been analyzed herein. The binding of MaRBC to human leukocytes strongly differs from the immune adherence phenomenon: rabbit anti-human CR1 did not react with MaRBC and the MaRBC receptor-binding activity is Ca2+-dependent. Rosette formation required intact energy metabolism and cytoskeleton integrity of leukocytes. Our attempts to purify the receptor from MaRBC membranes revealed the absence of CR1. Nevertheless, C3-binding proteins were isolated by selective desorption by Sepharose iC3 column chromatography. A three-band pattern was observed under reduced conditions with 74,000, 70,000, and 53,000 molecular weights. It was not possible to further separate these components. This protein complex inhibited the rosette phenomenon between MaRBC and both Raji and U-937 cells, exhibited a very poor cofactor activity, and had no decay-accelerating activity toward the classical C3 convertase. This material did not cross-react with antibodies directed to human proteins. These results showed that erythrocytes from new world monkeys do not express a receptor analogous to the human CR1, but expressed C3-binding protein with low cofactor activity that could recognize membrane-associated complement components.