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The interactions between proteins and gold colloids functionalized with protein-resistant oligo(ethylene glycol) (OEG) thiol, HS(CH2)11(OCH2CH2)6OMe (EG6OMe), in aqueous solution have been studied by small-angle X-ray scattering (SAXS) and UV-vis spectroscopy. The mean size, 2R, and the size distribution of the decorated gold colloids have been characterized by SAXS. The monolayer-protected gold colloids have no correlations due to the low volume fraction in solution and are stable in a wide range of temperatures (5-70 degrees C), pH (1.3-12.4), and ionic strength (0-1.0 M). In contrast, protein (bovine serum albumin) solutions with concentrations in the range of 60-200 mg/mL (4.6-14.5 vol %) show a pronounced correlation peak in SAXS, which results from the repulsive electrostatic interaction between charged proteins. These protein interactions show significant dependence on ionic strength, as would be expected for an electrostatic interaction (Zhang et al. J. Phys. Chem. B 2007, 111, 251). For a mixture of proteins and gold colloids, the protein-protein interaction changes little upon mixing with OEG-decorated gold colloids. In contrast, the colloid-colloid interaction is found to be strongly dependent on the protein concentration and the size of the colloid itself. Adding protein to a colloidal solution results in an attractive depletion interaction between functionalized gold colloids, and above a critical protein concentration, c*, the colloids form aggregates and flocculate. Adding salt to such mixtures enhances the depletion effect and decreases the critical protein concentration. The aggregation is a reversible process (i.e., diluting the solution leads to dissolution of aggregates). The results also indicate that the charge of the OEG self-assembled monolayer at a curved interface has a rather limited effect on the colloidal stabilization and the repulsive interaction with proteins.

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of mouse eggs, known as the zona pellucida. Over the past decade, powerful genetic, biophysical, and biochemical techniques have been employed to gain new insights into this interaction. Evidence from these studies does not support either of two major models for binding first proposed over two decades ago. Two more recently established models suggest that protein-protein interactions predominate during this initial stage of fertilization. Another model proposes that about 75-80% of the murine sperm bound to zona pellucida under well defined in vitro conditions is carbohydrate dependent, with the remaining sperm bound via protein-protein interactions. Mounting evidence suggests that the carbohydrate sequences coating the murine egg could be employed as specific immune recognition markers. Continued investigation of this system may resolve many of these controversial findings and reveal novel functions for murine zona pellucida glycoproteins.

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.

Objectives: CA125 expresses specific oligosaccharides that can inhibit the cytotoxicity of human natural killer (NK) cells. The current study was undertaken to determine the ability of CA125 to modulate NK cell-mediated cytotoxicity., Methods: CA125 was isolated from OVCAR-3 cells and its purity was determined by ELISA and ultra-sensitive mass spectrometric analysis. Peripheral blood-derived NK were treated with CA125 and standard cytotoxicity assays were performed using 51Cr-labeled K562 cells as targets. The expression of cell surface and intracellular markers on NK cells was determined by either flow cytometry or Western blot analysis., Results: NK cells incubated with CA125 for 72 h exhibited a 50-70% decrease in the lysis of K562 targets. Incubation with CA125 for 4 h and 24 h had no effect on NK-mediated cytolysis. Inhibition of NK function was observed at CA125 concentrations (10,000-100,000 U/ml) that are expected to be significantly lower than those observed in the tumor microenvironment. Co-stimulation with IL-2 did not abrogate the NK inhibitory response of CA125. CA125 did not reduce proliferation or induce apoptosis of NK cells and alter the expression of p56lck, phospholipase Cgamma1, ZAP70, or CD3zeta. CA125 did, however, induce major downregulation of CD16 and minor decrease in expression of CD94/NKG2A., Conclusions: Our ongoing research and recent work performed by other laboratories highlights the potential physiologic role of this mucin. Based on the data presented here, it is likely that the tumor-derived CA125 acts as a suppressor of the immune response that is directed against the ovarian tumors.

These guidelines about supervision, roles, and responsibilities are to assist in the appropriate utilization of occupational therapy personnel and in the appropriate and effective provision of occupational therapy services. All personnel are expected to meet applicable state and federal regulations, adhere to relevant workplace policies and the Occupational Therapy Code of Ethics (AOTA, 2000), and participate in ongoing professional development activities to maintain continuing competency.

Murine sperm initiate fertilization by binding to the outer covering of the egg known as the murine zona pellucida (mZP). This binding is thought to require the interaction of O-glycans linked to a specific mZP glycoprotein (mZP3) with egg-binding proteins coating the sperm plasma membrane. The precise molecular basis of this interaction remains to be resolved. In this study, we analyzed the O-glycosylation of the individual mZP glycoproteins by using ultrasensitive MS methods. We found that the majority of the O-glycans that are linked to mZP3 are core type 2 sequences terminated with sialic acid, lacNAc (Galbeta1-4GlcNAc), lacdiNAc (Gal-NAcbeta1-4GlcNAc), Galalpha1-3Gal, and NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4 (Sda antigen). Many of these terminal sequences have been implicated previously in murine sperm-egg binding. Core type 1 O-glycans are also present and are generally unmodified, although some are terminated with sialic acid, beta-linked N-acetylhexosamine, or NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4. Eggs expressing human ZP (huZP) glycoprotein huZP3, derived from transgenic mice, bind murine but not human sperm, implying that huZP3 acquires the same O-glycans as native mZP3. Sequencing of huZP3-associated O-glycans confirms that this implication is correct. The data obtained in this investigation may prove to be very useful for studies to determine the precise molecular basis of initial murine sperm-egg binding.

Based on the educational team recommendations, occupational therapists and occupational therapy assistants working in educationally related settings provide services to students who are eligible for Section 504 or special education under IDEA and need occupational therapy to benefit from their education program. It is the occupational therapist's responsibility to develop an intervention plan based on the student's needs and the therapist's professional knowledge base. The occupational therapist chooses and applies any frame of reference within the domain and process of occupational therapy. Regardless of the frame of reference utilized, the desired outcome of occupational therapy services is always engagement in occupations that allows participation in a student's daily life. When students demonstrate deficits in sensory integration that contribute to a significant and documented discrepancy in their skills within their educational program, the use of a sensory integrative approach may be one frame of reference for, intervention chosen by the occupational therapist.

CA125 is a mucin commonly employed as a diagnostic marker for epithelial ovarian cancer. Induction of humoral responses to CA125 leads to increased survival times in patients with this form of cancer, suggesting a potential role for this mucin in tumor progression. In this study, oligosaccharides linked to CA125 derived from the human ovarian tumor cell line OVCAR-3 were subjected to rigorous biophysical analysis. Sequencing of the O-glycans indicates the presence of both core type 1 and type 2 glycans. An unusual feature is the expression of branched core 1 antennae in the core type 2 glycans. CA125 is also N-glycosylated, expressing primarily high mannose and complex bisecting type N-linked glycans. High mannose type glycans include Man5-Man9GlcNAc2. The predominant N-glycans are the biantennary, triantennary, and tetraantennary bisecting type oligosaccharides. Remarkably, the N-glycosylation profiles of CA125 and the envelope glycoprotein gp120 (derived from H9 lymphoblastoid cells chronically infected with HIV-1) are very similar. The CA125-associated N-glycans have also recently been implicated in crucial recognition events involved in both the innate and adaptive arms of the cell-mediated immune response. CA125 may therefore induce specific immunomodulatory effects by employing its carbohydrate sequences as functional groups, thereby promoting tumor progression. Immunotherapy directed against CA125 may attenuate these immunosuppressive effects, leading to the prolonged survival of patients with this extremely serious form of cancer.

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.

We have previously suggested that the human fetus is protected during human development by a system of both soluble and cell surface associated glycoconjugates that utilize their carbohydrate sequences as functional groups to enable them to evoke tolerance. The proposed model has been referred to as the human fetoembryonic defense system hypothesis (hu-FEDS). In this paradigm, it has previously been proposed that similar oligosaccharides are used to mediate crucial recognition events required during both human sperm-egg binding and immune-inflammatory cell interactions. This vertical integration suggested to us that the sperm-egg binding itself is related to universal recognition events that occur between immune and inflammatory cells, except that in this case recognition of 'species' rather than recognition of 'self' is being manifested. In this paper, we have designated this component of hu-FEDS as the species recognition system (SRS). We propose that the SRS is an integral component of the hu-FEDS used to enable sperm-egg recognition and protection of the gametes from potential immune responses. Recent structural data indicates that the glycan sequences implicated in mediating murine gamete recognition are also expressed on CD45 in activated murine T lymphocytes and cytotoxic T lymphocytes. This overlap supports our contention that there is an overlap between the immune and gamete recognition systems. Therefore the hu-FEDS paradigm may be a subset of a larger model that also applies to other placental mammals. We therefore propose that the hu-FEDS model for protection should in the future be referred to as the eutherian fetoembryonic defense system hypothesis (eu-FEDS) to account for this extension. The possibility exists that the SRS component of eu-FEDS could predate eutherians and extend to all sexually reproducing organisms. Future investigation of the interactions between the immune and gamete recognition system will be required to determine the degree of overlap., (Copyright 2001 S. Karger AG, Basel)

Tamm-Horsfall glycoprotein (THP) is a major glycoprotein associated with human urine that binds pro-inflammatory cytokines and also inhibits in vitro T cell proliferation induced by specific antigens. THP derived from human pregnancy urine (designated uromodulin) has previously been shown to be 13-fold more effective as an inhibitor of antigen-induced T cell proliferation than THP obtained from other sources. Structural analysis of human THP and uromodulin has for the first time revealed that these glycoproteins are O-glycosylated. THP from nonpregnant females and males expresses primarily core 1 type O-glycans terminated with either sialic acid or fucose but not the sialyl Lewis(x) epitope. By contrast, the O-glycans linked to uromodulin include unusual core 2 type glycans terminated with one, two, or three sialyl Lewis(x) sequences. The specific association of these unusual carbohydrate sequences with uromodulin could explain its enhanced immunomodulatory effects compared with THP obtained from males and nonpregnant females. Analysis of THP from one of the pregnant females 2 months postpartum showed a reversion of the O-glycan profile to that found for a non-pregnant female. These data suggest that the glycosylation state of uromodulin could be under the regulation of steroidal hormones produced during pregnancy. The significant physiological implications of these observations are discussed.

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.

The recognition of carbohydrate epitopes by complimentary protein receptors has been shown to be a critical factor in gamete interaction in many different animal species. In this study it was hypothesized that, in the human, gamete binding requires an interaction between selectin ligands on the zona pellucida and putative egg binding proteins on the sperm surface. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of sperm to the zona) and advanced methods of carbohydrate analysis were used to test this hypothesis. From these tests it was shown that oligosaccharide recognition is also required for initial human gamete binding. This study suggests the existence of distinct egg binding proteins on human sperm that can bind to selectin ligands. Additionally, the results suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Glycoconjugates that manifest selectin-ligand activity and that express specific carbohydrate epitopes have potent contraceptive and immunosuppressive effects. Such specific oligosaccharide sequences may provide an appropriate recognition signal for embryo development and protection.

Glycodelins are glycoproteins synthesized in various glands, with sequence homology to beta-lactoglobulins, and named according to their unique oligosaccharide structures. We purified, cloned and sequenced endometrium- and seminal plasma-derived glycodelins (GdA and GdS respectively) and found that they are involved in various types of cell-cell communications. These include interactions between the spermatozoon and the egg, and between immune cells and their targets. Endometrial GdA inhibits sperm-egg binding, whereas the differently glycosylated GdS in seminal plasma does not. These observation are of interest for reproductive physiology, detection of causes of infertility, and they also may have potential for contraceptive development.

Protection of the gametes from potential immune responses is a primary function in human reproduction. The primary cell type responsible for the innate immune response in the uterus is the natural killer (NK) cell. NK cells normally recognize Class I major histocompatibility (MHC) molecules on potential target cells. Since both human spermatozoa and human oocytes do not express Class I MHC molecules on their surfaces, the appropriate cell surface signal that abrogates potential NK cell-mediated responses directed against these gametes is unknown. Recent evidence indicates that surface expression of bisecting-type N-linked glycans protects cells sensitive to NK cell-mediated lysis. We report that the zona pellucida of the human egg and plasma membranes of human spermatozoa potentially bind a lectin probe specific for bisecting type glycans in a carbohydrate-dependent manner. Since the innate immune response in the uterus is primarily mediated by NK cells, our results indicate that human gametes may be protected from this response by expressing bisecting type N-linked glycans on their surfaces.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycans from heparan sulfate proteoglycans on the cell surface of hepatocytes and is specifically cleared from the bloodstream by the liver. We show here that the two conserved regions, I and II-plus, of the CS protein, in a concerted action, preferentially bind to highly sulfated heparin-like oligosaccharides in heparan sulfate. In a concentration-dependent manner, peptides representing region I and region II-plus inhibited the binding of recombinant CS protein to HepG2 cells by 62 and 84%, respectively. Furthermore, the action of endoproteinase Arg-C, which cleaves the recombinant CS constructs CS27IVC and CSFZ(Cys) predominantly at the conserved region I, was inhibited by heparin in a concentration-dependent fashion. CSFZ(Cys), which has a higher affinity to HSPGs than CS27IVC, was stabilized by heparin at a w/w ratio (CS protein:glycosaminoglycan) of 20/1, whereas full protection of CS27IVC required more heparin (5/1). Heparan sulfate provided full protection of CSFZ(Cys) only at a ratio of 1/10. Native fucoidan as well as normally sulfated fuco-oligosaccharides (0.76 mol sulfate/mol fucose) inhibited Plasmodium berghei development in HepG2 cells by 84 and 66%, respectively, in a concentration-dependent manner and sporozoite invasion into CHO cells by 80%. Desulfated fucoidan oligosaccharides were inactive. These results may explain the selective interaction between the CS protein and the unique heparan sulfate from liver, which is noted for its unusually high degree of sulfation, and may provide a plausible explanation for the selective targeting of the malaria CS protein to the liver.

The primary molecular changes that lead to development of acquired immunodeficiency syndrome (AIDS) are very poorly understood, as are the mechanisms underlying the protection of the developing human from the maternal immune response. Recent data that the human immunodeficiency virus (HIV) may be using the glycosylation system of the T lymphocytes to acquire glycans for its glycoproteins that enable it to disrupt carbohydrate dependent immune cell interactions or induce aberrant immune reactions. Consistent with this hypothesis, gp120 from HIV infected human H9 lymphoblastoid cells expresses biantennary N-linked glycans with a bisecting GlcNAc sequence on 11% of their total oligosaccharides. This specific carbohydrate sequence has recently been shown to protect K562 erythroleukemic cells from natural killer (NK) cell responses when presented on the cell surface. We have recently demonstrated that bisecting biantennary type N-linked glycans are also expressed on the human zona pellucida (ZP); previous lectin binding studies indicate that is also expressed on human spermatozoa. Thus both the human gametes and HIV produced by H9 cells carry this same protective carbohydrate epitope on their outer surfaces. Human alpha-fetoprotein expressed in the developing human also carries the bisecting GlcNAc sequence, indicating that it may be suppressing the emerging fetal immune response by using its carbohydrate sequence as a functional group. We have suggested that the developing human and the gametes are also protected by soluble immunosuppressive glycoproteins found in the amniotic fluid and seminal plasma known as glycodelin-A (GdA) and glycodelin-S (GdS) respectively. Structural analysis of their N-linked oligosaccharides combined with other functional studies suggest that GdA and GdS employ their very unusual carbohydrate sequences as functional groups that enable them to manifest their immunosuppressive activities. GdA and GdS are significant components of our recently proposed model for the protection of the developing human and gametes designated the human fetoembryonic defence system hypothesis. A striking relationship now emerging is that the same unusual carbohydrate sequences associated with these immunosuppressive glycodelins are also specifically expressed on intravascular helminthic parasites, Helicobacter pylori, human tumour cells, and HIV infected T lymphocytes. The information presented in this review suggests that two new corollaries should be added to our recently proposed defence system hypothesis: (i) mimicry or acquisition of glycans that are used in this protective system by pathogens or tumour cells may enable them to either subvert or misdirect the human immune response, thereby greatly increasing their pathogenicity; and (ii) expression of glycoproteins used in this system by normal cells and tissues outside the reproductive system may protect them from immune responses, especially in those cases where major histocompatibility recognition is either absent or minimal. A better understanding of this hypothesis and its corollaries may enable us to address the molecular mechanisms underlying not only AIDS but also a host of other very serious pathological conditions in the human.

We have recently demonstrated that a human amniotic fluid-derived glycoprotein, glycodelin-A (GdA; previously known as PP14 or PAEP), potently inhibits gamete binding in an established sperm-egg binding system and expresses immunosuppressive activities directed against a variety of different immune cell types. GdA has high mannose-, hybrid-, and complex-type biantennary oligosaccharides including structures with fucosylated or sialylated N, N'-diacetyllactosediamine (GalNAcbeta1-4GlcNAc) sequences, which are rare in other human glycoproteins. We now report the characterization of glycodelin-S (GdS). This is a human seminal plasma glycoprotein that is immunologically indistinguishable from GdA, but unlike the latter, does not inhibit human sperm-zona pellucida binding under hemizona assay conditions. Analysis of the N-glycans of GdS by mass spectrometry revealed that all glycoforms of GdS are different from those of GdA. GdS glycans are unusually fucose-rich, and the major complex-type structures are biantennary glycans with Lewisx (Galbeta1-4(Fucalpha1-3)GlcNAc) and Lewisy (Fucalpha1-2Galbeta1-4(Fucalpha1-3)GlcNAc) antennae. It is probable that these highly fucosylated epitopes contribute to the immunosuppressive activity of human seminal plasma and to the low immunogenicity of sperm. This study provides the first evidence for gender-specific glycosylation that may serve to regulate key processes involved in human reproduction.

Glycodelin-A is a human amniotic fluid-derived glycoprotein with contraceptive and immunosuppressive activities. An immunoreactive form of glycodelin was detected in seminal plasma over a decade ago, but definitive characterization of this glycoprotein was not pursued. We considered it unlikely that the seminal plasma of fertile men would contain an appreciable amount of contraceptive glycodelin-A. To address this issue we purified seminal plasma glycodelin (glycodelin-S) and performed comparative studies with glycodelin-A. Glycodelin-S behaved differently when compared with glycodelin-A during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing but identically after enzymatic deglycosylation. N-terminal sequencing of glycodelin-A and glycodelin-S gave identical results, and digestion with trypsin gave identical peptide fragments. The glycoproteins were also found to be indistinguishable from each other based upon immunological analyses. These results indicate that glycodelin-S and glycodelin-A have similar overall protein structure, suggesting the likelihood that these glycoproteins are differentially glycosylated forms of very similar proteins. This latter possibility is supported by lectin binding studies indicating that, unlike glycodelin-A, glycodelin-S does not manifest any affinity for lectins from Wisteria floribunda or Sambucus nigra. The results of sugar analysis and neuraminidase digestion also lead us to conclude that glycodelin-S and glycodelin-A are differentially glycosylated forms of similar proteins. Our evidence indicates that glycodelin-A mediated its biological activities via its unusual oligosaccharide sequences that are not associated with glycodelin-S. In lectin-immunoassay no appreciable amount of contraceptive glycodelin-A was found in the 22 seminal plasma samples studied.

Educational practice is moving away from an "eligibility-oriented" student-problem approach toward a problem-solving approach that connects evaluation and interventions. This approach involves the identification of variables used to frame interventions, and, ultimately, it permits evaluation of intervention effectiveness. This article describes a problem-solving model developed and used within the Heartland Area Education Agency. This model relies on collecting baseline data and ongoing data, producing a basis not only for decision making, but also for evaluating the chosen intervention. This systematic and collaborative approach is used by the educational team to identify concerns about students' academic and nonacademic performance, to plan interventions, and to establish measurable outcomes.

The mechanisms underlying the protection of the human embryo/fetus from the maternal immune response are poorly understood. Substantial evidence indicates that carbohydrate recognition plays a primary role in the sequestration of leukocytes during inflammatory processes, lymphocyte homing, and initial gamete binding. Our previous studies suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Our more recent findings indicate that oligosaccharides participating in such processes are also associated with soluble glycoconjugates found in the human placenta, amniotic fluid, and decidua. We theorize that such glycoconjugates may abrogate the maternal immune/inflammatory response by blocking the primary adhesive interactions required for the expression of such activities. Foreign embryonic cells may also be protected by surface expression of oligosaccharide sequences that suppress immune effector cell action in a manner not dependent upon classical major histocompatibility (MHC) recognition. Glycoconjugates expressing selectin ligands may also manifest a potent contraceptive effect that may also be beneficial for both the mother and the developing embryo/fetus. This hypothesis provides a preliminary framework for understanding how temporally and spatially restricted immunosuppressive effects could be expressed in utero that protect the human embryo/fetus during this period of human development.

Glycodelin, also known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities. In this paper we report the first characterization of glycodelin-derived oligosaccharides. Using strategies based upon fast atom bombardment and electrospray mass spectrometry we have established that glycodelin is glycosylated at Asn-28 and Asn-63. The Asn-28 site carries high mannose, hybrid and complex-type structures, whereas the second site is exclusively occupied by complex-type glycans. The major non-reducing epitopes in the complex-type glycans are: Gal beta 1-4GlcNAc (lacNAc), GalNAc beta 1-4GlcNAc (lacdiNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacdiNAc), Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewisx), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (lacdiNAc analogue of Lewisx). It is possible that the oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae may manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell associated receptor. Oligosaccharides with fucosylated lacdiNAc antennae have previously been shown to potently block selectin-mediated adhesions and may perform the same function in glycodelin. The potent inhibitory effect of glycodelin on initial human sperm-zona pellucida binding is consistent with our previous suggestion that this cell adhesion event requires a selectin-like adhesion process. This result also raises the possibility that a convergence between immune and gamete recognition processes may have occurred in the types of carbohydrate ligands recognized in the human.

Binding of spermatozoa to the zona pellucida is an initial, crucial recognition event leading to fertilization. In the mouse, the best species characterized so far, the zona pellucida protein 3 (ZP3) has a central role as the specific, primary sperm receptor on the zona and as the inducer of the acrosome reaction. This sequence of events is not clearly understood as it relates to human gametes. The ideal test for the evaluation of sperm-zona pellucida interaction is one that can examine these events in a sequential fashion (i.e. binding followed by the acrosome reaction) in a standardized and specific bioassay. Here we have used the hemizona assay as an internally controlled test to examine human sperm-zona pellucida interaction. Results presented show that: (i) the hemizona assay has an excellent predictive power for IVF outcome and for the identification of male infertility; (ii) using a specific anti-ZP3 antiserum in immunocytochemical studies, the hemizona assay allows for the identification of structural/functional anomalies of the protein backbone of human ZP3 (identification of oocyte anomalies); and (iii) glycobiological studies using the hemizona assay model indicate that the initial sperm-zona pellucida binding requires a seletin-like interaction between the human gametes. These efforts may help us to characterize the cellular and molecular mechanisms involved in human gametes and their dysfunctions in infertile patients.

Fucoidan from Fucus vesiculosus inhibits human sperm-zona pellucida binding and blocks the zona pellucida-induced acrosome reaction in human sperm. Fucoidan also potently inhibits selectin-mediated adhesion of leukocytes to vascular endothelium. To understand the molecular basis for fucoidan's inhibition of specific cell adhesion events, we have investigated the structure of this fucan using definitive methods of carbohydrate structural analysis. We report the revised average structure for fucoidan. [formula: see text] This average structure differs from the previous model for fucoidan in two respects. First, the core region of the fucan is composed primarily of a polymer of alpha 1-3-linked fucose with sulfate groups substituted at the 4 position on some of the fucose residues. Secondly, fucose is also attached to this polymer to form branch points, one for every 2-3 fucose residues within the chain. This revised average structure is consistent with previous studies suggesting a branched random coil as the best model for this polysaccharide. The proposed model is also a closer structural analogue of the sulfated carbohydrate ligands that bind to selectins. This information should be useful for determining whether a relationship exists between selectin-mediated adhesion of leukocytes and human sperm-egg binding.

We previously reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits: (1) tight binding of human sperm to human zona pellucida in vitro and (2) stimulation of the acrosome reaction by acid solubilized human zona pellucida. Here, we determined fucoidin binding activity on human spermatozoa and its localization on both live and permeabilized human sperm populations. A typical binding curve was demonstrated with biotinylated fucoidin. In competitive inhibition assays with unlabelled fucoidin or human sperm membrane extracts, IC50's were 4.0 micrograms/ml and 31.4 micrograms/ml, respectively. Fucoidin binding was localized over the acrosomal region of methanol-fixed human sperm and this pattern of binding significantly decreased from 92 +/- 3% to 74 +/- 6% with calcium ionophore A23187 treatment (p < 0.01). Binding of fucoidin-coated beads to live (non-permeabilized) human sperm was less than 1%. Addition of the detergent, Triton-X, to permeabilize sperm membranes resulted in a significant increase in binding (p = 0.001). These results provide evidence for the presence of a fucoidin binding compound in human spermatozoa that is localized to the membranes of the acrosomal region and can be extracted by a mild detergent extraction. Absence of binding by fucoidin to intact but not permeabilized spermatozoa suggests that the heteropolysaccharide binds to a receptor within the acrosomal matrix. However, further investigation is warranted to determine whether a fucoidin binding site is present both at the sperm's surface for the initial contact with the zona pellucida, and also for secondary binding after exposure of the acrosomal membranes.

The authors recently reported that fucoidin (a polymer of predominantly L-fucose sulfate) produced a strong, significant, and dose-dependent inhibition of sperm-zona binding under hemizona assay conditions. The current studies were designed to evaluate the mechanisms underlying this inhibitory activity. Using computerized semen analysis, the monoclonal anti-sperm antibody T-6 and indirect immunofluorescence technique, and the fura-2 indicator, no significant impact of fucoidin on sperm motion parameters, on the spontaneous acrosome reaction, or its prerequisite, the increase in calcium influx, were observed. Subsequently, a mild acid hydrolysis of the fucoidin molecule was performed, followed by sizing hydrolysates in a Biogel P2 column and separating fractions (n = 6). All fucoidin fragments significantly inhibited tight binding of human sperm to human zona pellucida under hemizona assay conditions (range, 56%-94%) when tested individually. These results provide further evidence that the effect of fucoidin is produced by a receptor-ligand association.

We have metabolically labeled the human pancreatic tumor cell line PANC-1 with high specific activity tritiated sugar precursors to study the expression of glycosphingolipids by this cell type. We have used a combination of detergent solubilization, exhaustive protease digestion, ceramide glycanase digestion, and reverse-phase chromatography to isolate glycosphingolipid-derived oligosaccharides specifically labeled in their component sugars. A significant proportion of the oligosaccharides derived from polar glycosphingolipids were of high molecular mass (greater than 2000 Da). The results of compositional studies, lectin affinity chromatography, and methylation analysis suggested that this high molecular weight fraction consists of lactosaminoglycan type oligosaccharides derived from polyglycosylceramides. There are on average three beta 1-6 linked N-acetyllactosamine branches attached to the polylactosamine backbone in this type of glycosphingolipid-derived oligosaccharide. The majority of the oligosaccharides also contain 1-2 mol of sialic acid that are linked alpha 2-3 to penultimate galactose. The results indicate that PANC-1 cells, like human colorectal tumor cells, express highly extended neolacto type glycosphingolipids. However, the lactosaminoglycan sequences are highly branched, unlike those associated with colorectal tumor cells.

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.

Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.

Fucoidin and heparin sulfate inhibit binding of human sperm to the human zona pellucida under hemizona assay (HZA) conditions. Here we used the HZA to further assess tight sperm binding with/without preincubation of the sperm with other sulfated and nonsulfated glycoconjugates and charged polymers. Fucoidin significantly inhibited binding compared with controls (greater than 75% inhibition), even if sperm were washed after preincubation with the saccharide. Dextran sulfate also produced significant inhibition, although to a lesser extent (54% inhibition). Chondroitin sulfates A and B, heparin, and dextran did not affect binding. Sodium sulfate and polyglutamic acid did not affect HZA results; polyphosphates produced only moderate inhibition. The potent inhibitory effect of the sulfated carbohydrates fucoidin and dextran is probably competitive (receptor-ligand type) in nature. However, the lack of significant effects of simple charged molecules (nonspecific effects) suggests that the degree of sulfation (charge) may not be crucial to its inhibitory action.

Diagnostic tests for Actinobacillus pleuropneumoniae have been problematic because current tests do not use a purified antigen and in most cases measure either antibody or antigen, but not both. We describe a Farr-type double-label radioimmunoassay that utilizes purified, serotype-specific, 3H-capsule to measure antibody to capsule directly or that can measure capsule in a sample indirectly by inhibition of antibody binding. The assay could detect about 1 ng of serotype-specific antibody in serum or at least 100 pg of capsule in a sample. Due to the sensitivity of the assay, false-positive results were common with neat sera (probably due to cross-reacting antibodies to unrelated antigens), but the specificity was improved when the sera were diluted 1:100. The radioimmunoassay should prove to be a useful reference method for research and diagnostic testing and for comparison of new assays for detection of capsule or antibodies to capsule.

The postnatal development of mammalian skeletal muscle is characterized by changes in the properties of several key membrane glycoprotein enzymes and receptors. In the present study, CMP-sialic acid: fetuin sialyltransferase and CMP-sialic acid: lactosylceramide sialyltransferase activity was characterized in sarcolemma and sarcoplasmic reticulum membranes isolated from neonatal (0-1 week) and adult (8 week) rabbit skeletal muscle. CMP-sialic acid: fetuin sialyltransferase decreased by a factor of 10 in sarcolemma and 6 in sarcoplasmic reticulum during development, whereas CMP-sialic acid: lactosylceramide sialyltransferase activity decreased by a factor of 6 in sarcolemma and 18 in sarcoplasmic reticulum. The Km for CMP-sialic acid using the lipid acceptor declined during the development of sarcoplasmic reticulum (neonate vs. adult: 538 vs. 33 microM), but not in sarcolemma. The carbohydrate composition of sarcolemma was changed only with respect to total sialic acid content (neonate vs. adult: 67 vs. 44 nmol/mg). Similar analysis of sarcoplasmic reticulum carbohydrates showed decreases in total sialic acid, lipid-bound sialic acid, hexosamines and hexoses. The major ganglioside was GM3 for both types of membrane. No qualitative changes were observed in ganglioside composition comparing neonatal and adult membranes.

Thirty mouse monoclonal antibodies (MoAbs) raised against human sperm detect common antigenic determinants on human lymphocytes, erythrocytes, bacteria and endotoxin. Specific chemical, enzymatic and lectin blocking studies indicate that the sperm-associated antigens defined by these MoAbs are glycoconjugates. Further studies including reactivity of these MoAbs with organic sperm extracts indicate that the predominant carriers of these carbohydrate antigens are glycolipids and that the terminal immunodominant monosaccharide may be N-acetyl-glucosamine or N-acetylgalactosamine.

This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin. This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.

The postnatal development of skeletal muscle is characterized by changes in membrane function associated with N-linked glycoproteins. In the present study, early reactions involved in the synthesis of the dolichol-linked core oligosaccharide were examined in neonatal and adult rabbit skeletal muscle sarcoplasmic reticulum membranes. The initial rate of N-acetylglucosamine incorporation in the presence of exogenous dolichol phosphate was similar between neonate and adult (3.5-4.1 pmol of GlcNAc/min/mg). The Km values for UDP-GlcNAc and exogenous dolichol phosphate were similar. Tunicamycin (0.04-0.08 micrograms/ml) inhibited N-acetylglucosamine incorporation by 50%. UDP-GlcNAc pyrophosphatase activity was greater in neonatal membranes than adult (840 versus 350 pmol of GlcNAc-1-P/min/mg), explaining, in part, the greater enhancement of neonatal GlcNAc incorporation by pyrophosphatase inhibitors. Nucleotide-sugar pyrophosphatase inhibitors (alpha, beta-methylene ATP and dimercaptopropanol) increased the capacity of neonatal activity 4-fold and adult enzyme 2-fold. Analysis of dolichol-linked products by mild acid hydrolysis however, revealed that neonate had higher capacity for N,N'-diacetylchitobiosyl(pyro)phosphoryldolichol synthesis than adult. Mannosyltransferase and glucosyltransferase were elevated 6- and 5-fold in neonate compared to adult membranes. Neonate exhibited 4-fold greater GDP-Man pyrophosphatase activity than adult (500 versus 125 pmol of Man-1-P/min/mg). The Km for GDP-Man increased in the presence of exogenous dolichol phosphate. Increasing concentrations of exogenous dolichol phosphate did not equalize neonate and adult mannosyltransferase activity, indicating that the decline in activity during development was not due to a decrease in a pool of dolichol phosphate accessible to mannosyltransferase. Glucosyltransferase for the synthesis of glucosylphosphoryldolichol was also elevated 5-fold in neonatal compared to adult sarcoplasmic reticulum (7 versus 1.4 pmol of Glc/min/mg). In a previous study, it was reported that glycoprotein sialyltransferase activity decreased by a factor of 6.5 during the postnatal maturation and that total membrane hexose content of sarcoplasmic reticulum decreased by a factor of 8. Together, these results suggest that the postnatal development of skeletal muscle is characterized by coordinated changes in the expression of enzymes involved in both the "early" and "late" reactions of N-linked oligosaccharide biosynthesis.

We examined the interaction between immobilized wheat germ agglutinin and the large, polylactosamine-containing glycans from human erythrocytes and human K-562 erythroleukemic cells. Three classes of interaction were identified. One class of glycan was merely retarded during chromatography. The other two classes were retained and could be distinguished by their ease of displacement with N-acetylglucosamine (GlcNAc); one was a moderate-affinity fraction displaced by 0.1 M GlcNAc and the other was a high-affinity fraction subsequently displaced by 1.0 M GlcNAc. A relatively small fraction of the K-562 polylactosamines were in the high-affinity class. We explored the role that fucose and sialic acid substitutions play in the strength of the lectin-glycan interaction. Although sialic acid is recognized by wheat germ agglutinin, sialylation was not required for the high-affinity interaction, and the presence of sialic acids actually prevented some glycans from binding with high affinity. In contrast, fucose is not part of the binding determinant, yet the removal of fucose resulted in decreased affinity. The possibility that some of these changes in affinity were the result of conformational changes was explored using matrices that had wheat germ agglutinin immobilized at different densities. At low wheat germ agglutinin densities, adult and fetal erythroglycans and K-562 glycophorin-like glycans were not retained by the matrix. As the density increased, the proportion of glycans that were retarded, and ultimately retained, increased. While these increases in the proportions retained occurred in parallel for the three different glycans, the apparent affinities of the glycan-lectin interactions differed. The glycophorin-like glycans were always readily displaced by 0.1 M GlcNAc, even at higher wheat germ agglutinin densities. In contrast, as the wheat germ agglutinin density increased, the proportion of erythroglycans that could be displaced by 0.1 M GlcNAc decreased; at 10 mg/ml immobilized wheat germ agglutinin, greater than 80% of the erythroglycans exhibited this tighter interaction. We suggest that this higher affinity interaction is the result of the large glycans spanning adjacent wheat germ agglutinin molecules, and is determined by the proximity of these molecules and the conformation of the glycans.

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.