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Objective: We generated induced pluripotent stem (iPS) cells from a patient with distal myopathy with rimmed vacuoles (DMRV), in which sialic acids synthesis is reported to be defective. In this study, we examined whether the differentiation to retinal pigment epithelial (RPE) cells and autophagy was affected in the patient derived cells., Methods: Patient derived iPS cells were established through the transduction of re-programming factors into peripheral mononuclear cells via retrovirus vectors. RPE cells were induced from iPS cells through aggregation culture. Then the autophagy induced by amino acid starvation was estimated by measuring LC3-containing "puncta" structure., Results: A 3D aggregate culture of patient-derived iPS cells resulted in some irregular shapes, and the aggregate contained large vacuoles filled with lipid droplets and cellular components such as damaged mitochondria. RPE cells induced from patient-derived iPS cells showed impaired autophagy flux under amino acid starvation., Conclusion: These findings were similar to those of sialidosis patient-derived iPS cells, in which cleavage of terminal sialic acids in oligosaccharide chains is defective. This suggests that the control of both the addition and removal of sialic acids are pivotal for autophagy progression.

Antidepressant drugs are too often ineffective, the exact mechanism of efficacy is still ambiguous, and there has been a paucity of novel targets for pharmacotherapy. In an attempt to understand the pathogenesis of depression and subsequently develop more efficacious antidepressant drugs, multiple theories have been proposed, including the modulation of neurotransmission, the upregulation of neurogenesis and neurotrophic factors, normalizing hypothalamic-pituitary-adrenal reactivity, and the reduction of neuroinflammation; all of which have supporting lines of evidence. Therefore, an ideal molecular target for novel pharmaceutical intervention would function at the confluence of these theories. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) functions broadly, serving to mediate synaptic plasticity, neurogenesis, neurotrophic factor signaling, and inflammatory signaling throughout the brain; all of which are associated with the pathophysiology and treatment of depression. Moreover, the expression of PSA-NCAM is reduced by depression, and conversely enhanced by antidepressant treatment, particularly within the hippocampus. Here we demonstrate that selectively cleaving the polysialic acid moiety, using the bacteriophage-derived enzyme endoneuraminidase N, completely inhibits the antidepressant efficacy of the selective-serotonin reuptake inhibitor fluoxetine (FLX) in a chronic unpredictable stress model of depression. We also observe a corresponding attenuation of FLX-induced hippocampal neuroplasticity, including decreased hippocampal neurogenesis, synaptic density, and neural activation. These data indicate that PSA-NCAM-mediated neuroplasticity is necessary for antidepressant action; therefore PSA-NCAM represents an interesting, and novel, target for pharmacotherapy.

Chronic immobilization stress (CIS) shortens apical dendritic trees of CA3 pyramidal neurons in the hippocampus of the male rat, and dendritic length may be a determinant of vulnerability to stress. Expression of the polysialylated form of neural cell adhesion molecule (PSA-NCAM) in the hippocampal formation is increased by stress, while PSA removal by Endo-neuraminidase-N (endo-N) is known to cause the mossy fibers to defasciculate and synapse ectopically in their CA3 target area. We show here that enzymatic removal of PSA produced a remarkable expansion of dendritic arbors of CA3 pyramidal neurons, with a lesser effect in CA1. This expansion eclipsed the CIS-induced shortening of CA3 dendrites, with the expanded dendrites of both no-stress-endo-N and CIS-endo-N rats being longer than those in no-stress-control rats and much longer than those in CIS-control rats. As predicted by the hypothesis that endo-N-induced dendritic expansion might increase vulnerability to excitotoxic challenge, systemic injection with kainic acid, showed markedly increased neuronal degeneration, as assessed by fluorojade B histochemistry, in rats that had been treated with endo-N compared to vehicle-treated rats throughout the entire hippocampal formation. PSA removal also exacerbated the CIS-induced reduction in body weight and abolished effects of CIS on NPY and NR2B mRNA levels. These findings support the hypothesis that CA3 arbor plasticity plays a protective role during prolonged stress and clarify the role of PSA-NCAM in stress-induced dendritic plasticity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cells generated in the subventricular zone give rise to neuroblasts that migrate to the olfactory bulb (OB) along the rostral migratory stream (RMS). The polysialylated form of neural cell adhesion molecule (PSA-NCAM) is expressed by these cells, and has been shown to both promote their migration and suppress differentiation induced by NCAM. In the present study, enzymatic removal of PSA from these neuroblasts using PSA-specific endoneuraminidase has been found not only to disrupt the tangential migration and cellular organization of the RMS, but also to cause a massive dispersion of BrdU (5-bromo-2'-deoxyuridine)-labeled neuroblasts into surrounding brain regions, including cortex and striatum. These dispersed cells are capable of differentiation, some into mature neurons, and could potentially be of value in the repair of CNS injury. Although the removal of PSA by genetic deletion of NCAM also results in a smaller OB and a swollen RMS, the cells do not escape the RMS in large numbers. These findings suggest that the presence of NCAM without PSA plays a role in the dispersion process, possibly by inducing a new pattern of migration associated with NCAM-dependent differentiation.

The unique modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is tightly associated with nervous system development and plasticity. The prevailing view that this large carbohydrate polymer acts as an anti-adhesive factor seems straightforward at first sight. However, during almost 25 years of polySia research it became increasingly clear that the impact of polySia on cell surface interactions can not be explained by one unifying mechanism. Recent progress in the generation of mouse models, which partially or completely lack polySia due to ablation of one or both of the two polySia synthesizing enzymes, provides novel insights into the function of this unique post-translational modification. The present review is focused on a phenotype comparison between the newly established mouse strains which combine polySia-deficiency with normal NCAM expression and the well-characterized NCAM negative mouse model. Analysis of shared and individual phenotypes allows a clear distinction between NCAM and polySia functions and revealed that polySia plays a vital role as a specific control element of NCAM-mediated interactions.

The neural cell adhesion molecule (NCAM) has been implicated in regulating synaptic plasticity mechanisms as well as memory consolidation processes. Attachment of polysialic acid to NCAM (PSA-NCAM) has been reported to down-regulate its adhesive forces, a process hypothesized to be implicated in synapse selection after learning experiences. PSA-NCAM has been critically implicated in hippocampus-related synaptic plasticity and memory storage, but information about its functional role in other brain areas remains scarce. Here, we studied mice deficient for polysialyltransferase-1 (ST8SialV/PST-1), an enzyme which attaches PSA to NCAM during postnatal development and adulthood, and whose deficiency results in a drastic reduction of PSA-NCAM expression throughout the brain in adulthood. Mice were tested for their performance in the water maze and auditory fear conditioning (AFC). We report that ST8SiaIV knockout mice were impaired in spatial as well as reversal learning in the water maze. On the other hand, AFC was intact and ST8SiaIV mice exhibited no impairments in the acquisition or retention of cued fear memories. Spatial orientation learning and reversal learning require complex integration of spatial information and response selection involving the hippocampus and prefrontal cortex, whereas cued fear conditioning is an associative type of emotional memory that highly depends on amygdala function. Therefore, our results indicate that PSA-NCAM contributes differentially to learning processes that differ in the nature of the neural computations involved, which probably reflects a differential role of this molecule in different brain regions.

Carbohydrate-deficient transferrin (CDT) measurements are considered a reliable marker for chronic alcohol consumption, and its use is becoming extensive in forensic medicine. However, CDT is not a single molecular entity but refers to a group of sialic acid-deficient transferrin isoforms from mono- to trisialotransferrin. Thus, the development of methods to analyze accurately and precisely individual transferrin isoforms in biological fluids such as serum is of increasing importance. The present work illustrates the use of ICPMS isotope dilution analysis for the quantification of transferrin isoforms once saturated with iron and separated by anion exchange chromatography (Mono Q 5/50) using a mobile phase consisting of a gradient of ammonium acetate (0-250 mM) in 25 mM Tris-acetic acid (pH 6.5). Species-specific and species-unspecific spikes have been explored. In the first part of the study, the use of postcolumn addition of a solution of 200 ng mL(-1) isotopically enriched iron (57Fe, 95%) in 25 mM sodium citrate/citric acid (pH 4) permitted the quantification of individual sialoforms of transferrin (from S2 to S5) in human serum samples of healthy individuals as well as alcoholic patients. Second, the species-specific spike method was performed by synthesizing an isotopically enriched standard of saturated transferrin (saturated with 57Fe). The characterization of the spike was performed by postcolumn reverse isotope dilution analysis (this is, by postcolumn addition of a solution of 200 ng mL(-1) natural iron in sodium citrate/citric acid of pH 4). Also, the stability of the transferrin spike was tested during one week with negligible species transformation. Finally, the enriched transferrin was used to quantify the individual isoforms in the same serum samples obtaining results comparative to those of postcolumn isotope dilution and to those previously published in the literature, demonstrating the suitability of both strategies for quantitative transferrin isoform determination in real samples.

Polysialic acid (PSA) regulates functions of the neural cell adhesion molecule (NCAM) during development and in neuroplasticity in the adult; the underlying mechanisms at different phases of learning and memory consolidation are, however, unknown. To investigate the contributions of PSA versus the extracellular domain of the NCAM glycoprotein backbone to synaptic plasticity, we applied NCAM, PSA-NCAM, and PSA to acute slices of the hippocampal CA1 region of NCAM-deficient mice and measured their effects on long-term potentiation (LTP). Remarkably, only PSA and PSA-NCAM, but not NCAM restored normal LTP. Application of these molecules to the dorsal hippocampus of wild-type mice showed that PSA-NCAM and PSA, but not NCAM, injected before fear conditioning, impaired formation of hippocampus-dependent contextual memory. Consolidation of contextual memory was affected by PSA-NCAM only when injected during its late, but not early phases. None of the tested compounds disturbed extrahippocampal-cued memory. Mice lacking the polysialyltransferase (ST8SialV/PST) responsible for attachment of PSA to NCAM in adulthood showed a mild deficit only in hippocampal contextual learning, when compared with NCAM-deficient mice that were disturbed in both contextual and cued memories. Contextual and tone memory in NCAM-deficient mice could be partially restored by injection of PSA-NCAM, but not of NCAM, into the hippocampus, suggesting that the impact of PSA-NCAM in synaptic plasticity and learning is not mediated by modulation of NCAM-NCAM homophilic interactions. In conclusion, our data support the view that polysialylated NCAM is involved in both formation and late consolidation of contextual memory.

Under conditions of strong secretion of neurohypophysial hormone, such as during parturition, lactation and dehydration, the hypothalamic oxytocin-system displays a remarkable morphological plasticity such that astrocytic coverage of its neurones diminishes, their surfaces become directly juxtaposed and contacted by an increased number of synapses. A growing body of evidence indicates that these anatomical changes have an impact on glutamatergic neurotransmission in the supraoptic nucleus, and may be therefore of physiological consequence. We here evaluated the consequences of the inhibition of such plasticity on the overall activity of the oxytocin system during lactation. Remodeling was prevented by performing hypothalamic microinjections in gestating rats of endoneuraminidase, an enzyme that removes polysialic acid from the neural cell adhesion molecule. Our earlier studies established that the presence of polysialic acid is a prerequisite for remodeling of the oxytocin system in the supraoptic and paraventricular nuclei. In dams in which polysialic acid was absent in all magnocellular nuclei after bilateral endoneuraminidase injections, parturition was normal and neither the frequency nor the amplitude of suckling-induced reflex milk ejections was different from vehicle-treated dams. The weight gain of pups was also normal as was water intake by the dams. We then assessed the electrical activity of antidromically identified magnocellular neurones in the polysialic acid-free supraoptic nucleus of isoflurane-anesthetized lactating rats. Basal and bursting activity characteristic of oxytocin neurones before each reflex milk ejection was not significantly different from that recorded in the supraoptic nucleus of rats with normal levels of polysialic acid. Our results indicate that neuro-glial remodeling, despite its role on fine modulation of oxytocin neuronal activity, is not essential to parturition and lactation.

Under chronic conditions of neuropathic pain, nociceptive C terminals are lost from their target region in spinal lamina II, leading to reduced thermal hyperalgesia. This region of the spinal cord expresses high levels of polysialic acid (PSA), a cell surface carbohydrate known to weaken cell-cell interactions and promote plasticity. Experimental removal of PSA from the spinal cord exacerbates hyperalgesia and results in retention of C terminals, whereas it has no effect on plasticity of touch Abeta fibers and allodynia. We propose that expression of PSA at this stress pathway relay point could serve to protect central circuitry from chronic sensory overload.

Polysialylated neural cell adhesion molecule (PSA-NCAM) is predominantly expressed during development where it regulates biological functions including axon targeting and neuronal precursor cell migration. Although dramatically down regulated after birth in most regions of the nervous system, PSA-NCAM remains highly expressed into adulthood in areas that have ongoing regeneration and plasticity such as in the olfactory bulb and hippocampus. Consequently, lack of PSA-NCAM in NCAM null mice results in distinct morphological changes to these areas. The functional correlates of these changes are not well defined although there have been reports that learning is impaired in NCAM null mice. In the present study, we assessed the ability of old and young NCAM null mice to learn an odour discrimination task. We tested male and female experimental and control animals of two different ages: 30-60 days and 12-15 months. During 4 days of training, NCAM null and C57BL/6J received trials where one odour (CS+) was paired with sugar while another odour (CS-) was not. In a subsequent preference test, conducted in the absence of sugar, all animals, regardless of strain or age, spent significantly more time digging in the CS+ odour than in the CS- odour. In addition, there was no significant difference in digging behaviour in the CS+ between the NCAM null and the control animals. These data indicate that deletion of the NCAM gene may change the morphology of the olfactory bulb but does not interfere with the ability to learn an odour discrimination task., (Copyright 2003 Elsevier B.V.)

Developing chick retinotectal projections extend rostrally in the superficial stratum opticum of the tectum until they reach their appropriate target zone. They then penetrate, arborize, and form synapses within distinct tectal retinorecipient layers. In this study, we show that the polysialylated neural cell adhesion molecule is expressed both on the membrane of these developing projections and in the stratum opticum and retinorecipient layers during the period of optic innervation. On this basis, the role of polysialic acid was analyzed with respect to both trajectory and arborization in the tectum, using confocal imaging of DiI-labeled retinotectal fibers in whole-mount tecta of embryos pretreated with a polysialic acid-specific degrading enzyme, endoneuraminidase N. The removal of polysialic acid caused several distinct abnormalities, including random dorsal/ventral meandering of fibers in the stratum opticum, a distorted branching and extension of arbors in the retinorecipient layers, and inappropriate synaptic vesicle accumulation in pretarget areas. These findings indicate that the unique ability of polysialic acid to regulate different types of cell interactions is an essential component of axon behavior during multiple steps of tectal target innervation., (Copyright 2003 Wiley-Liss, Inc.)

Using the model of lysolecithin-induced demyelination of the corpus callosum in wild-type, NCAM-deficient, and endoneuraminidase-injected mice, we have analyzed the consequences of the loss of expression of NCAM or PSA residues on the migration and proliferation capacities of neural precursors of the subventricular zone (SVZ). We showed that the absence of PSA or NCAM delayed migration of neural precursors to the olfactory bulb and consequently enhanced their recruitment at the lesion site. Moreover, after demyelination, the lack of NCAM but not PSA promoted proliferation in the SVZ and the lesion while the lack of PSA favored the differentiation of the traced cells into the oligodendroglial fate both in the SVZ and in the lesion. As previously demonstrated in vitro (L. Decker et al., 2000, Mol. Cell. Neurosci. 16, 422-439), these data illustrate the involvement of PSA and NCAM in neural precursor motility and differentiation in the normal and injured central nervous system, suggesting distinct roles for these two molecules under pathophysiological conditions., ((C)2002 Elsevier Science (USA).)

Galactosialidosis is a rare lysosomal storage disease associated with deficiencies of alpha-galactosidase and beta-neurominidase. In this report, two siblings with galactosialidosis, resembling Niemann-Pick disease with the presence of foamy cells in multiple organs, splenomegaly and prominent hepatomegaly, are presented. Galactosidase deficiency and an increased number of urinary sialic acid compounds were determined in these cases, and prenatal diagnosis was performed for their fourth sibling. Besides the presence of the typical clinical features, enzyme study is essential for the diagnosis of lysosomal storage disease and it facilitates in making the prenatal diagnosis.

Serum carbohydrate-deficient-transferrin (CDT) was measured by a micro anion-exchange chromatography/enzyme immunoassay. Results obtained on 245 sera analyzed in four laboratories were compared. Moreover, one laboratory used a commercial kit with ready-to-use microcolumns and a radioimmunoassay for measuring eluted CDT. Imprecision was judged to be satisfactory. Within-assay coefficients of variation ranged from 5 to 10%, between-assay coefficients of variation ranged from 9 to 18%. Between-laboratory results were compared for 110 sera from control subjects (daily alcohol intake < 40 g), for 57 sera from chronic ethylic subjects and for 78 sera from patients suffering from non-alcoholic liver diseases. There was a large between-laboratory variation, suggesting that the method is difficult to standardize and that results are not transferable. Results of enzyme and radioimmunoassays were compared on 325 sera. The best correlation was obtained in the groups of ethylic subjects and those with non-alcoholic hepatic diseases. Finally the performance of the CDT-test was evaluated by calculating sensitivity and specificity. With both methods specificity was very high (> 85%) but sensitivity was poor (< 50%).

Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this 'artificial' Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc alpha- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.

Chronical alcohol ingestion may induce conformational molecular modifications of plasma transferrin: alcohol modifies the content of its carbohydrates. The abnormal transferrin contains reduced amounts of carbohydrates, especially sialic acid, constituting its terminal trisaccharides biantennary chains. Plasma levels of partly deficient or asialotransferrin increase in chronically drinkers. In English speaking countries, it is called carbohydrate deficient transferrin or CDT. A positive correlation is obtained between the plasmatic concentration of CDT and the amount of ingested alcohol. Positivity and sensitivity of CDT are superior to those other usual biological parameters. The CDT quantitation may be proposed for the detection and the follow-up of alcohol drinkers, in order to evaluate the degree of intoxication, and during the period of withdrawal.

Two brothers presented with olivopontocerebellar atrophy of neonatal onset. The clinical features (failure to thrive, hypotonia, liver disease, effusions, and visual inattention) were similar to those of the four cases already reported, as were the necropsy findings of olivopontocerebellar atrophy, hepatic steatosis and fibrosis, and microcystic renal changes. The clinical similarities between this and the disialotransferrin developmental deficiency syndrome were noted. The characteristic abnormality of serum transferrin found in the latter syndrome was also found in the two cases reported here. We suggest that both syndromes are caused by the same, or related, defects in glycoprotein metabolism.

We report on five girls (including monozygotic twins) with a newly recognized disease comprising severe neurologic disturbances, variable hepatomegaly, abnormal subcutaneous fat distribution and skeletal anomalies. The neurologic picture was characterized by moderate to severe psychomotor retardation, alternating internal strabismus , hypotonia, hyporeflexia and ataxia. Biochemical investigations showed a number of abnormalities such as tubular proteinuria, slightly increased serum transaminases, hypoalbuminemia, hypo-beta-lipoproteinemia and decreased serum thyroxine-binding globulin. Moreover there was retinitis pigmentosa, cerebellar hypotrophy and electrophysiologic evidence for a peripheral neuropathy. However, histologic examination of a nerve biopsy in one of the patients failed to show myelin abnormalities. On the other hand, abnormal lamellar inclusions were found in the lysosomes of some Schwann cells and of liver tissue as well. Additional investigations in four patients revealed a deficiency of sialic acid, galactose and N-acetylglucosamine of plasma glycoproteins. Enzymatic analysis of serum suggested a deficiency of an N-acetyl-glucosaminyltransferase. Remarkably, the (healthy) fathers but not the mothers presented the same carbohydrate deficiencies of plasma glycoproteins albeit to a much lesser degree. The mode of hereditary transmission of this disease remains unclear; the possibility of X-linked inheritance is under investigation.

Bernard-Soulier syndrome is a constitutional thrombopathy with an impaired platelet adhesion to the vessel wall. Since the first description in 1948 many works had been reported, and recently a molecular abnormality of the platelet membrane was shown. Interactions between specific membrane sites and platelet functions are now proposed.

The spleen of the exhypoxic polycythemic mouse was employed to study the effect of erythropoietin on the synthesis of chromosomal proteins. At 1, 3, 18, 36, 45, and 72 hr after injection of erythropoietin, spleens were removed, minced, and incubated with 3H-arginine for 1 hr. Chromatin was isolated from the labeled splenic tissue and separated into histone and nonhistone protein (NHP) fractions. An increase in the incorporation of 3H-arginine into NHP occurred within 3 hr and into histones by 18 hr. Incorporation of 3H-arginine into both histones and NHP was maximal by 45 hr and had declined by 72 hr. Total histone specific activity increased fivefold while NHP specific activity increased twofold. Histones and NHP were fractionated on polyacrylamide gels and a double isotope labeling proceudre was used to study the synthesis of the individual histone proteins and NHP. Following administration of erythropoietin, there was a coordinate increase in the specific activity of all five major histone proteins and most of the NHP. The earliest change in specific activity of both histones and NHP occurred prior to the appearance of morphologically identifiable erythroblasts in the spleen.

The factor VIII/von Willebrand factor (f.VIII/vWf) protein was purified from the plasma of a patient with von Willebrand's disease (vWd). The patient had all of the classic laboratory findings of vWd except for the ristocetin-induced platelet aggregation of his own platelet-rich plasma. The disease has been documented in three generations. Comparison of the purified normal and vWd f.VIIi/vWf protein revealed several abnormalities, including decreased concentration of f.VIII/vWf antigen; decreased specific vWf activity; absence of the larger molecular forms of the f.VIII/vWf protein; carbohydrate deficiencies affecting the sialic acid, penultimate galactose and N-acetylglucosamine moieties; and decreased binding of the f.VIII/vWf protein to its platelet receptor. These studies indicate the multiplicity of biochemical and functional abnormalities associated with the f.VIII/vWf protein in vWd. f.VIII/vWf protein to normal f.VIII/vWf protein that had been treated with 2-mercaptoethanol (2-ME) to reduce the multimer size and then treated with specific exoglycosidases to remove the sialic acid and penultimate galactose residues revealed similar biologic properties.

A reduction of red cell SA in patients following acute myocardial infarction is reported and the effects of SA-depleted red cells on cardiac index and alveolar capillary blood flow in the dog are described. The mean red cell SA in 26 patients following acute myocardial infarction was 0.021 +/- 0.001 compared with a mean of 0.031 +/- 0.002 mumol./0.1 ml RBC in 12 normal subjects (p less than 0.01). In five dogs injected with neuraminidase, an enzyme which removes SA from the red cell membrane, a 43% decrease in mean cardiac index from 2.3 +/- 0.22 to 1.3 +/- 0.16 (p less than 0.01) occurred. In films of the pulmonary microcircuation the mean widths of typical alveolar capillary beds decreased 42.6% +/- 5% (p less than 0.01). In three other dogs, autotransfusion with SA-depleted stored blood resulted in a 25% decrease in mean cardiac index from 2.0 +/- 0.21 to 1.5 +/- 0.21 (p less than 0.2), and a 21.7% +/- 0.9% (p less than 0.01) decrease in mean widths of typical alveolar capillary beds. We conclude that a reduction of red cell SA follows acute myocardial infarction and that SA-depleted red cells decrease cardiac index and alveolar capillary blood flow in the dogs.

In 1969, it was shown that platelets of patients with Bernard-Soulier (B-S) syndrome exhibited an altered electrophoretic mobility arising from a surface membrane sialic acid deficiency. Subsequent studies showed that a major membrane glycoprotein, GP Ib, was either deleted, reduced in concentration, or structurally modified in B-S platelets. Controversy persists, however, as to the specificity of the defect in platelets of different patients, especially with regard to studies performed on patients in different laboratories. We have now studied platelets from six patients, and during these studies have analyzed the platelet proteins and glycoproteins by single and two-dimensional SDS-PAGE or by crossed immunoelectrophoresis. The surface proteins of the platelets of different patients have been radiolabelled with 3H or 125I. Our studies point to the deletion or severe reduction in concentration of GPIb alpha and Ib beta in the platelets of five patients, detectable but reduced levels of Ib were present in the platelets of the other patient. Studies using 3H-labelled glycoproteins suggest that mol.wt. 82,000 (GP V) and mol.wt. 17,000 glycoproteins are also missing or abnormal. The relationship between these additional defects and the major GP Ib lesion remains to be determined. It should be emphasized that these are membrane abnormalities, no alpha-granule or cyto-plasmic protein deficiencies have been located in B-S platelets.

A covalently coupled IgG-latex reagent was instrumental in isolating rheumatoid factors (RF) from normal immunoglobulins in the sera of patients with rheumatoid arthritis. It was found that RF contained hyposialylated immunoglobulins of both IgG and IgM classes. This biochemical alteration might be relevant to understanding the etiopathogenesis of the disease.

A total of 39 samples of hostile mucus, as defined by postcoital examination, were examined for N-acetylneuraminic acid (NANA) deficiency, as measured by the enzymatic addition of NANA, spermatozoal penetration and immobilization. Only 56.7% of the mucus samples were deficient in NANA and this did not correlate with spermatozoal penetration or immobilization, which were negatively correlated. Thus, as the spermatozoal hostility in the mucus decreases, spermatozoal penetration increases. This finding also applies to hostile mucus not deficient in NANA. In contrast, resialylation of NANA hostile mucus, deficient and not deficient in NANA, although not enhancing spermatozoal penetration, did reduce spermatozoal immobilization. Thus, components of the mucus deficient in NANA and/or the lack of unbound NANA may contribute to mucus hostility, but it is not the only hostile factor. In addition, SEM studies of NANA-deficient mucin before and after resialylation were shown to have similar structures. Hence ultrastructural changes are not apparent in NANA-deficient mucin, and this supports the previous finding that NANA deficiency does not impede spermatozoal penetration. The spermatozoa from the husbands of the infertile couples formed three distinct groups in terms of spermatozoal penetration and immobilization in normal donor mucus. One group demonstrated normal levels of spermatozoal penetration and immobilization in donor mucus. A second group was demonstrable in which spermatozoal penetration was similar to that in the wife's hostile mucus, but had a normal level of spermatozoal immobilization. In the third group, both spermatozoal penetration and immobilization in donor mucus were similar to that in the wife's hostile mucus. The results demonstrate that not all hostile mucus is deficient in NANA, and that other unknown factors are involved. In addition, there are also male factors which may impede spermatozoal penetration and/or result in the inability of the spermatozoa to survive in normal donor mucus.