Confirmation of the Cholinergic Specificity of the Chol-1 Gangliosides in Mammalian Brain Using Affinity-Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus

An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cholinergic-specific immune lysis of mammalian brain synaptosomes and recognizes a group of minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyltransferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

RAPID SCREENING FOR SALMONELLA IN FISHMEAL BY ENRICHMENT-IMMUNOASSAY

We previously described an enrichment-immunoassay utilizing a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Here we evaluated it for the rapid screening for Salmonella in fishmeal obtained from the national Animal and Plant Quarantine service in the People's Republic of China. In this method, the number of Salmonella present is first expanded by appropriate enrichment cultures, and the pathogens are then directly detected by the T6 immunoassay. In a total of 94 enrichment cultures of fishmeal, we obtained an overall concordance of 98% between the results obtained in parallel by this method and by conventional test method. The positive prediction by this method was 92% and the negative prediction was 100%. The turn around time for the new test was 27 h which is a significant improvement from the turn around time exceeding 96 h required for the conventional test method. This test proved to be compatible with the routine work flow in the practical setting of a quarantine laboratory.     

Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells

Background  

Mannose binding lectin (MBL) is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro.     

Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons

Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.     

Nerve Growth Factor Enhances the Expression of the Cholinergic-Specific Ganglioside Chol-1 in Cocultures of Rat Septum and Hippocampus

Cocultures of septal and hippocampal tissues taken from 6- to 7-day-old rats were maintained in culture for up to 30 days in the presence and absence of nerve growth factor (NGF), and their Chol-1 contents determined at varying time intervals by a modified enzyme-linked immunosorption assay (ELISA). The major brain gangliosides were determined densitometrically after spraying chromatograms with resorcinol reagent. There was little change in the contribution of the major gangliosides to the total ganglioside content of the explants either with time or the presence or absence of NGF, the only exception being an NGF-insensitive fall in the contribution of GM1 to about 60% of its initial value at 20 and 30 days. By contrast, the concentration of Chol-1 expressed either per unit weight of ganglioside sialic acid or protein increased considerably in culture and this increase was enhanced by NGF. The effect of NGF resembles that on other cholinergic markers, choline acetyltransferase and acetylcholinesterase, and may be attributed to an NGF-stimulated hippocampal ingrowth of cholinergic fibres and enhanced survival of cholinergic septal neurons. The Chol-1 concentration finally attained in the presence of NGF and the time course of its increase parallel those previously found in vivo and indicate the potential usefulness of septal-hippocampal cocultures for investigating the function of Chol-1.