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Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in "bundles" and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae close to mineralized "islands" in the cartilage and hypertrophic chondrocytes containing accumulated matrix proteins. Immunostaining revealed: (1) that types I, II, VI and X collagens and aggrecans were deposited intracellulary and (2) co-localization within the islands of types I, II, X collagens and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non-conclusive, only single nucleotide polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification., (© 2010 John Wiley & Sons A/S.)

Rapport et al. (11) reported that marked aberrations in brain ganglioside profiles were present in 17 of 21 patients with ALS. The aberrations were detected both in motor cortex and in unexpected regions such as frontal, temporal, and parahippocampal gyrus cortex. These results suggest that some underlying pathological process in ALS also occurs in some neurons that are less vulnerable than motor neurons to consequent deterioration. Since gangliosides are major membrane constituents whose carbohydrate residues establish structural configurations on the external face of the cell membrane, it is highly probable that aberrant ganglioside patterns reflect alterations in receptor structure and function. Receptors are inherently cell specific and the specificity would account for differences in response of sensory and motor neurons to the pathological process in ALS. An apparent absence of similar ganglioside aberrations in spinal cord suggests that the primary pathology is in the brain. Such aberrations are not seen in Alzheimer's disease. If receptor functions are altered in ALS, what ligands might be involved? A major consideration is neurotrophic hormones (2). Gangliosides are known to modulate the effect of nerve growth factor in some in vitro systems and very recent evidence implicates protein kinase activation as an important mechanism.

Analysis of the antigenic components of synaptic membranes presents several problems caused by detergents required to solubilize the proteins. These problems involve detection of the reaction between antibodies and proteins in the detergent extract as well as characterization of the individual antigens after further purification. We found a satisfactory solution for analysis of rat synaptic membranes by using rocket immunoelectrophoresis with an intermediate gel containing 0.5% berol to sequester sodium dodecyl sulfate (SDS) and an antibody gel containing 0.1% berol to maintain the solubility of the proteins. With antiserum prepared against a highly purified synaptic membrane fraction, these conditions gave rocket heights proportional to protein concentration (for the major rocket) and showed no reactions with membrane extracts of liver, kidney and spleen. By separation on gradient slab gels containing SDS, 4 antigenic polypeptides were found in Berol extracts of synaptic membrane, having apparent molecular sizes of 56,000, 58,000, 62,000 and 66,000 daltons. The SDS extract of the berol insoluble residue contained 3 antigenic polypeptides (12,500; 15,000, 16,500 daltons). The combined use of gradient slab gel electrophoresis in SDS and rocket immunoelectrophoresis with an intermediate gel to remove SDS provides a sensitive and rapid method for definitive identification of individual antigenic components in detergent extracts of membrane and for further purification of these antigens.

The effect of antibodies to GM1 ganglioside on release of neurotransmitters from rat brain slices was studied. Depolarization-induced (40 mM-KCl or veratrine) release of gamma-aminobutyric acid was markedly enhanced. Depolarization-induced release of norepinephrine was only slightly enhanced, whereas that of serotonin was unaffected. No effect on spontaneous release was observed for any of these three neurotransmitters. These results show that antibodies that can bind to synaptic membrane antigens may alter neurotransmitter release and that antibodies directed against GM1 ganglioside exhibit a measure of specificity in producing such an effect.

Topical cortical injection into rats of antiserum to the synaptic membrane fraction caused recurrent spiking activity as observed in the EEG; neither antiserum to S-100 protein nor antiserum to myelin caused any such abnormality. These same rats, when observed on a maze learning task (Lashley III) responded differently to the different antisera. Rats receiving injections of antiserum to the synaptic membrane fraction or antiserum to S-100 protein were inhibited in their performance, while rats injected with antiserum to myelin behaved like uninjected animals.

Two-month-old male offspring of rats injected intravenously on day 19 of gestation with antiserum to synaptic membrane fraction showed marked behavioral deficits on a DRL (differential reinforcement at low rates) training paradigm. These behavioral deficits included perseveration, slow acquisition rates, and poor retention. Offspring of pregnant rats injected with antiserum to galactocerebroside or isotonic saline did not show these behavioral effects.

The sequence of events leading to the formulation of the aldehydogenic chain in plasmalogens as an alpha, beta-unsaturated ether is described.

In a search for evidence of biochemical disorders in regions of postmortem brain other than the motor cortex in amyotrophic lateral sclerosis (ALS), ganglioside patterns were also examined in the frontal, temporal, and parahippocampal gyrus cortex. In 21 ALS brains studied (20 sporadic, 1 familial), abnormal patterns were found in the frontal cortex (81%), temporal cortex (75%), motor cortex (70%), and parahippocampal gyrus cortex (71%). Patterns were established by measuring the percentage distribution of 12 ganglioside species. Two abnormal patterns were detected. One was based on low proportions of GD1b, GT1b, and GQ1b associated with high proportions of GM2 and GD3 (GM1, GD1a, GD2, and GT1a values were normal). The second abnormality was the appearance of Gx. Neither abnormality was seen in the 13 non-ALS control brains. The first, and predominant, abnormality was found in the frontal cortex in 14 brains, and the second was observed in 13 brains; 10 brains showed both abnormalities. These findings thus constitute evidence that the disease process in ALS extends beyond the motor cortex and involves neurons in several brain areas.

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.

Antisera against rat erythrocytes contain agglutinins directed against unknown lipid determinants. Complement-fixation shows more reactivity with lipid extracts of rat spleen than of other rat tissues. The isolation of the reactive lipid from rat spleen, cytolipin S, is described. Cytolipin S is a glycosphingolipid containing glucose, galactose, and galactosamine with molar ratios of 1:2:1. It migrates on TLC like asialo GM1 (more slowly than cytolipin R, a ceramide tetrasaccharide, or cytolipin F, a ceramide pentasaccharide). Asialo GM1 and cytolipin S, when properly combined with auxiliary lipids, react very similarly with anti-rat erythrocyte sera by complement-fixation. However, cytolipin S is much more effective than asialo GM1 in inhibiting the hemagglutination reactions. It is concluded that cytolipin S and asialo GM1 are ceramide tetrasaccharides having different chemical structures and that the structural differences probably will be found in the carbohydrate linkages other than that between the terminal and penultimate residues.

Following observations that the intracerebral injection into rats of antiserum to brain gangliosides resulted in recurrent epileptiform activity and that seizure activity was not seen if antibodies were removed by absorption of the antiserum with pure GM1 ganglioside, a study was undertaken to establish characteristics of the immunological agents used to produce this model of epilepsy. It was determined that the potencies (antibody titers with GM1 ganglioside) of antiganglioside sera can be correlated with the intensities of epileptiform activity they induce; that immunoglobulin fractions from antiganglioside sera are even more effective biologically than the antisera; and that antibodies to GM1 ganglioside purified by affinity chromatography can also induce recurrent epileptiform discharges but are not as effective as either native antiserum or immunoglobulin fractions.

Antibodies to GM1 ganglioside injected into the sensorimotor cortex of the rat induce recurrent epileptiform activity. We now find that the divalent F(ab')2 and monovalent Fab' fragments derived from antiganglioside IgG molecules are able to induce epileptiform seizures. This result supports the view that the binding of antibodies to ganglioside receptors in the synaptic membrane is sufficient in itself to initiate changes in membrane processes which lead to epileptiform spiking. These changes do not appear to be dependent on linking of ganglioside receptors or on the presence of serum factors such as complement.

The subcellular distribution in rat brain cortex of six synaptic membrane antigens (56K, 58K, 62K, 63K, 64K, 66K) was studied by rocket immunoelectrophoresis, using antiserum to a highly purified synaptic plasma membrane fraction. Initial analysis of the insoluble portion of subcellular fractions showed that these antigens were also present in smooth microsomes, rough microsomes, and synaptic vesicles; that only traces were present in synaptic junctions; and that none was present in nuclei, mitochondria, and myelin. A trace amount of activity was also present in synaptic vesicle cytosol, but none in whole brain cytosol. Quantitative measurements of synaptic plasma membranes, smooth microsomes, and synaptic vesicles showed that all six antigens were present in synaptic plasma membranes and smooth microsomes, but that the 66K antigen was absent from synaptic vesicles. The 56K, 58K, 62, 63K, and 64K antigens were present in highest concentration in synaptic plasma membranes, whereas the 66K antigen content was highest in smooth microsomes. Only the 58K, 62K, and 63K antigen were detectable in the membrane fraction of whole brain. Their enrichments in synaptic plasma membranes were 10.9, 5.4, and 5.9, respectively. We conclude that the 56K, 58K, 62K, 63K and 64K antigens are primary components of synaptic plasma membranes. The presence of synaptic plasma membrane antigens in smooth microsomes and synaptic vesicles probably represents material being actively transported, consistent with the hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are transported via smooth endoplasmic reticulum.

Five-day-old rats received a single injection (50 microliter) of antiserum to ganglioside into the cisterna magna and were compared to control animals injected with the antiserum which had been absorbed with pure GM1 ganglioside to remove the specific antibodies. Both groups showed normal rates of body growth. However, animals receiving antiganglioside serum had, at 60 days of age, impaired performance when tested on a complex learning task (DRL) as well as chemical and morphological alterations in the somatosensory cerebral cortex. Gross morphology and wet weight of whole brain were normal in both groups. Microchemical analysis of somatosensory isocortex revealed a normal content of total solids, protein, and DNA. However, ganglioside sialic acid, galactocerebroside, and RNA were decreased by 31%, 32% and 25% of control values, respectively (P less than 0.01). Quantitative measurements of oblique dendrites of Golgi-stained cortical pyramidal neurons revealed a 31% decrease in the number of spines. Additionally, the majority of spines were of the stubby configuration, whereas dendrites from controls were populated predominately by thin spines. These observations suggest that antibodies to GM1 ganglioside interfere with optimal neonatal development on both dendrites and myelin in cerebral cortex. The results provide an animal model in which an immunologically-mediated disturbance of cortical development is associated with chronic behavioral impairment.

Glycoproteins in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups by galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components were fractionated on diatrizoate to give four synaptosomal membrane fractions (A to D) and a mitochondrial pellet (E). Fractions A and B represent highly purified synaptosomal plasma membranes. After separation of their polypeptides by electrophoresis, 4/5 of the label was present in two bands: one about 72 000 and the other between 7800 and 3200 daltons. Seven other bands were labeled to various degrees: 160 000, 96 000, 53 000, 39 000, 34 000, 23 000 and 16 000 daltons. With isolated membranes (which incorporate 5--6 times more label) 4/5 of label was present in polypeptides in three ranges: 160 000--96 000, 70 000--40 000 and 7800--3200. The number of polypeptides that can be labeled by treatment of isolated membranes is very large. In comparison, glycoproteins whose topographical distribution permits interaction with large molecules at the synpatic surface are very limited. It is further suggested that the external synaptosome membrane involves a relatively tight network of interacting molecules that cannot be readily penetrated by large molecules.

The binding of serotonin to a soluble, high affinity binding protein (SBP) present in synaptosomes and assoicated with serotonergic tracts, has now been studied for the effect of drugs and, in particular, drugs interacting with contractile proteins. Vincristine, vinblastine, and cytochalasin B were found to cause 50% inhibition of serotonin binding to SBP at 1.5 X 10(-6)M, 7.5 X 10(-6)M and 50 X 10(-6)M, respectively. Colchicine did not affect the binding at 1 X 10(-3)M. When vinblastine was injected intraventricularly, the binding capacity of SBP isolated from brain 20 and 26 hours after injection was decreased 42% and 60% respectively. It is concluded that SBP is an actin-like contractile protein, distinct from microtubulin, and not associated with membranes. The storage of serotonin through binding to a protein with such properties suggests that this compartment is in some way involved in the translocation or transfer of serotonin from one part of the cell to another.

The immunocytochemical localization of glial fibrillary acidic portein within glioma cell bodies and their processes by the immunoperoxidase method is demonstrated to be of diagnostic value. This method has advantages over "special" stains because it is not so dependent upon color alone, and because it identifies a specific protein in the cells. The immunoperoxidase method using antiserum to glial fibrillary acidic protein is shown to be useful for the differentiation of mixed glial and mesenchymal tumors, and for the diagnosis of tumors in which a glial or mesenchymal cell origin is in doubt.

A single injection of 10 microliters of antiserum to total brain ganglioside onto (and into) the sensorimotor cortex of the rat resulted in recurrent spiking in the cortical electroencephalogram, lasting from 7 to 17 days. Absorption of antibody with pure monosialoganglioside (GM1) completely abosished the effect. Spiking was reactivated after 4 weeks by intramuscular injection of pentylenetetrazole.

GM1 ganglioside injections (i.p.) reduce amphetamine-induced asymmetric rotation in rats 48 h after a partial unilateral transection of the nigrostriatal pathway. We found that this reduction was maximal when rats received their first GM1 injection within 2 h after surgery. Rats injected 4-12 h after surgery, or rats only pretreated with GM1, showed no significant effect on rotation. Striatal membrane Na+,K+-ATPase in rats injected with GM1 0-2 h after hemitransection showed only a 10% loss in activity (versus the untransected hemisphere) as compared to control losses of 38%. The maintenance of membrane Na+,K+-ATPase activity in GM1-treated rats may be one mechanism by which a balance between hemispheres in striatal dopaminergic transmission is preserved, resulting in reduced asymmetric rotation. The observation that there is a critical postsurgical period when GM1 administration results in optimal functional recovery supports our hypothesis that gangliosides are exerting an acute effect on damaged CNS tissue. This acute effect is further evidenced by the reduced loss of membrane Na+,K+-ATPase following injury.

Eleven monoclonal antibodies to GM1 ganglioside were prepared from hybridoma clones obtained by fusion of spleen cells from mice immunized with GM1 with mouse myeloma cells. When the reactivities of these 11 monoclonal antibodies were determined by enzyme-linked immunosorbent assay with six glycosphingolipids (GM1, GD1a, GD1b, GT1b, GM2, and asialo-GM1), they showed different degrees of specificity. From their reactivity patterns, they could be divided into three groups: Group 1, those that react only with GM1 (C3 and D3); Group 2, those that react predominantly with GM1 (C6, B6, D1, e1, g1, g9, and e12); and Group 3, those that show poor discrimination (h2 and A4). The clones differed in their biological activities.

The monosialoganglioside GM1 can potentiate the neuritogenic activity of media conditioned by several cell types: neonatal glia, C6 glioma, embryonic chick heart or skeletal muscle and the rat myogenic line L6. To probe further the neuritogenic activity of conditioned media (CM), 5 mouse monoclonal antibodies (mAbs) against GM1, designated B6, C3, C4h2, D1 and D3 were incorporated individually into nutrient medium (NM) supplemented with CM prior to incubation with sensory ganglia. Nine-day embryonic chick dorsal root ganglia were explanted onto collagen-coated coverslips and incubated at 35 degrees C for 5 h in NM supplemented with 150 micrograms/ml GM1. After washing with NM, the explants were re-fed with NM + CM containing 20% mAb and cultured for an additional 43 h. The resultant neuritogenesis was evaluated microscopically by determining mean neurite number and length of randomly mixed cultures. The 5 antibodies differed in their capacities to inhibit CM-mediated neuritogenesis of these primed target cells. D1 and D3 were most effective in reducing neurite length and number produced by all sources of the CM, while C3 and C4h2 were intermediate in their inhibition of neurite initiation (number). The effect of B6 on neurite initiation and elongation was the least. The ability of these mAbs to inhibit neuritogenic activity of CM derived from both glial and myogenic tissue suggests that gangliosides play a basic role in neuronal development. The differing responses elicited by the individual mAbs may reflect a relationship between the structural complexity of the GM1 molecule and the neuritogenic mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)