Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Cyclosporine A treatment in four cases of aplastic anemia

Cyclosporine A (CyA) treatment of 4 patients with severe aplastic anemia, who were ineligible for bone marrow transplantation, was carried out for periods of between 12 weeks to 20 months. A normalization of Hb and bone marrow, together with a marked improvement in WBC and platelet counts, were observed in only one of these four patients. The remission was maintained for 20 months under continuous treatment. A relapse occurred only when the patient himself interrupted treatment. No serious side effects were observed with CyA doses of 4-10 mg/kg/daily and blood concentrations of 200-400 ng/ml. No significant changes in T helper/T suppressor ratios were noted during the course of CyA treatment.     

Factors Affecting Competence for Transformation in Staphylococcus aureus

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage phi11 can be transformed. Superinfection of competent cells with phi11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower.     

Ethanol and glycerol production under aerobic conditions by wild-type,respiratory-deficient mutants and a fusion product of Saccharomyces cerevisiae

Summary Experiments were performed to investigate growth, ethanol and glycerol production by wild-type strains (RHO) and respiratory-deficient (rho) mutants of Saccharomyces cerevisiae. Furthermore protoplasts were fused in order to enhance the fermentation capacity of a flocculent strain. At high substrate conditions, 150 g/l of saccharose, there is no difference in cell growth. However, at a glucose concentration of 10–20 g/l the mutants grow much slower. After 3 days of incubation at 28° C in a complete medium the viability of the two strains is the same. In minimal medium on the other hand the number of viable cells of the mutant is 100-fold reduced. All mutants tested showed a higher specific activity of alcohol dehydrogenase (ADH I) and an enhanced production of glycerol compared with the wild-type strain. By protoplast fusion a modified flocculent strain was obtained with higher specific activity of ADH I and a reduced biosynthesis of glycerol. However, the yields of ethanol (75–78%) are about the same for the wild-type strain and the rho mutants under aerobic conditions in absence of catabolite repression.     

Isolation and partial purification of the major abundant class rat seminal vesicle poly(A+)-messenger RNA

Total poly(A(+))-RNA (poly(A(+))-RNA(tot)) was isolated from rat seminal vesicle and its size distribution determined by 70% formamide 5-25% sucrose density analysis. One major peak was resolved in the 10-13 S region and accounted for approximately 35% of the total poly(A(+))-RNA applied. Preparative 1% SDS, 5-20% linear sucrose density gradients also resolved a single major peak in the 11S region (poly(A(+))(11S). Analysis of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) under denaturing conditions on 2% agarose gel electrophoresis demonstrated two major components in both poly(A(+))-RNA populations. Size estimations for these components are 620 and 540 NT respectively. (3)H-cDNA was made to both poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S). Back-hybridization of poly(A(+))-RNA(tot) and poly(A(+))-RNA(11S) to their respective (3)H-cDNA revealed a highly abundant class representing 41% and 85% of the sequences in their respective (3)H-cDNA's. The highly abundant class corresponded to 3-5 sequences present in 30,000-50,000 copies/cell. Invitro translation of poly(A(+))-RNA(11S) resulted in two major polypeptides coded for by the 620 NT long and 540 NT long poly(A(+))-RNA respectively.Images     

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUNDThe development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIMTo investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs).METHODSSeven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle.RESULTSFollow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones.CONCLUSIONOur data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.     

Antibodies to Heteromeric Glycolipid Complexes in Guillain-Barré Syndrome

Autoantibodies are infrequently detected in the sera of patients with the demyelinating form of Guillain-Barré syndrome most commonly encountered in the Western world, despite abundant circumstantial evidence suggesting their existence. We hypothesised that antibody specificities reliant on the cis interactions of neighbouring membrane glycolipids could explain this discrepancy, and would not have been detected by traditional serological assays using highly purified preparations of single gangliosides. To assess the frequency of glycolipid complex antibodies in a Western European cohort of patients GBS we used a newly developed combinatorial glycoarray methodology to screen against large range of antigens (11 gangliosides, 8 other single glycolipids and 162 heterodimeric glycolipid complexes). Serum samples of 181 patients from a geographically defined, Western European cohort of GBS cases were analysed, along with 161 control sera. Serum IgG binding to single gangliosides was observed in 80.0% of axonal GBS cases, but in only 11.8% of cases with demyelinating electrophysiology. The inclusion of glycolipid complexes increased the positivity rate in demyelinating disease to 62.4%. There were 40 antigens with statistically significantly increased binding intensities in GBS as compared to healthy control sera. Of these, 7 complex antigens and 1 single ganglioside also produced statistically significantly increased binding intensities in GBS versus neurological disease controls. The detection of antibodies against specific complexes was associated with particular clinical features including disease severity, requirement for mechanical ventilation, and axonal electrophysiology. This study demonstrates that while antibodies against single gangliosides are often found in cases with axonal-type electrophysiology, antibodies against glycolipid complexes predominate in cases with demyelinating electrophysiology, providing a more robust serum biomarker than has ever been previously available for such cases. This work confirms the activation of the humoral immune system in the dysimmune disease process in GBS, and correlates patterns of antigen recognition with different clinical features.     

Expression of the Gm1-Species, [NeuN]-GM1, in a Case of Human Glioma

Altered glycosylation is a common feature in tumors of various kind and particular interest has been focused on the expression of tumor-associated gangliosides. We have previously identified some human glioma-associated gangliosides and in this study yet another, not previously described, ganglioside has been isolated. The ganglioside was prepared from human glioma tissue taken at autopsy. The new ganglioside bound cholera-toxin B-subunit and its structure was confirmed by fast atom bombardment—mass spectrometry to be NeuN-GM1 (II³NeuNH₂-GgOse₄Cer). In the dissected tumor specimen, the concentration of NeuN-GM1 was 0.1 mol/g wet weight and accounted for approximately 20% of the monosialoganglioside fraction. Normal human brain tissue specimens (n = 10) did not contain detectable (>0.5 nmol/g wet weight of tissue) amounts of NeuN-GM1, indicating that this ganglioside might be associated with human glioma. However, none of the 17 other tumour specimens reveal any detectable amounts of this ganglioside. In conclusion, NeuN GM1 is a glioma-associated ganglioside but its exceptional expression limits its relevance as a molecule involved in general tumor biology.     

Gene therapy: prospects for glycolipid storage diseases

Lysosomal storage diseases comprise a group of about 40 disorders, which in most cases are due to the deficiency of a lysosomal enzyme. Since lysosomal enzymes are involved in the degradation of various compounds, the diseases can be further subdivided according to which pathway is affected. Thus, enzyme deficiencies in the degradation pathway of glycosaminoglycans cause mucopolysaccharidosis, and deficiencies affecting glycopeptides cause glycoproteinosis. In glycolipid storage diseases enzymes are deficient that are involved in the degradation of sphingolipids. Mouse models are available for most of these diseases, and some of these mouse models have been used to study the applicability of in vivo gene therapy. We review the rationale for gene therapy in lysosomal disorders and present data, in particular, about trials in an animal model of metachromatic leukodystrophy. The data of these trials are compared with those obtained with animal models of other lysosomal diseases.     

Sulfatide is associated with insulin granules and located to microdomains of a cultured beta cell line

Previous studies using pancreas from various mammals and freshly isolated islets from rat pancreas have provided evidence supporting possible involvement of the glycosphingolipid sulfatide in insulin processing and secretion. In this study, sulfatide expression and metabolism in the beta cell line RINr1046-38 (RIN-38), commonly used as a model for beta cell functional studies, were investigated and compared with previous findings from freshly isolated islets. RIN-38 cells expressed similar amounts (2.7 +/- 1.1 nmol/mg protein, n = 19) of sulfatide as isolated rat islets and also followed the same metabolic pathway, mainly through recycling. Moreover, in agreement with findings in isolated islets, the major species of sulfatide isolated from RIN-38 cells contained C16:0 and C24:0 fatty acids. By applying subcellular isolations and electron microscopy and immunocytochemistry techniques, sulfatide was shown to be located to the secretory granules, the plasma membrane and enriched in detergent insoluble microdomains. In the electron microscopy studies, Sulph I staining was also associated with mitochondria and villi structures. In conclusion, RIN-38 cells might be an appropriate model, as a complement to isolated islets where the amount of material often limits the experiments, to further explore the role of sulfatide in insulin secretion and signal transduction of beta cells.