Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.      

Characterization of liposome suspensions by flow cytometry

Novel approaches to drug delivery and induction of immune responses using liposomes have received much attention in recent years. Liposomes, however, are not a singular entity, but can be produced with a diverse group of phospholipids that form microspheres of different sizes, physical structure, electrochemical characteristics, and most importantly, physiologic properties. The purpose of this study was to establish the usefulness of flow cytometry as a convenient, rapid method for assessing the relative size and uniformity of liposomal preparations. Liposomes were made from phospholipid suspensions by sonication alone, or sonication followed by microemulsification. Forward laser light scatter (FSC) analysis of liposomal preparations by flow cytometry indicated that microemulsification produced homogeneous, small vesicles which were less than 1 micron in diameter, compared to the more heterogeneous sized liposomes generated by sonication alone. Transmission electron micrographs of the liposomal preparations were used to confirm the FSC results and showed that liposomes prepared by microemulsification were homogeneous, unilamellar vesicles which exhibited a mean diameter of 99.8 nm, whereas the sonicated-only preparation was more heterogeneous in size, exhibiting a mean diameter of 154.1 nm. Analysis of various liposome preparations by FSC during a 9 week storage period showed that small vesicles were relatively stable. We conclude that flow cytometry using FSC analysis provides a rapid, reproducible and convenient method to evaluate the relative size, uniformity and stability of liposomes.     

Phosphoinositide deficiency due to inositol depletion is not a mechanism of lithium action in brain

The "inositol depletion hypothesis" has been widely held to be the explanation for both the effect of lithium on brain function, apropos of its use in mood disorders, and on the impairment of development and induction of embryonic malformations in diverse organisms. The essence of the hypothesis is that a deficiency in cellular myo-inositol (Ins), secondary to lithium inhibition of inositol monophosphatase and/or multiple inositol polyphosphate phosphatase activities with trapping of Ins as inositol phosphates, leads to a depression of phosphatidylinositol (PtdIns) and a secondary impairment in inositide signaling. However, the ability of relatively low micromolar levels of Ins to reduce mammalian PtdIns synthetase activity in vivo has never been adequately tested. We have generated a lethal murine brain Ins deficiency model and measured PtdIns content using a novel MALDI-TOF MS method. Our results show that in the most severe Ins deficiency ever recorded in a mammal, the brain PtdIns levels do not decrease. We conclude that PtdIns deficiency due to "inositol depletion" is not a mechanism of lithium action in brain, and that Ins plays another unidentified role in the mammalian brain.     

The conditioning effect of optic nerve injury upon axonal regrowth from adult rat retinal ganglion cells explanted in vitro

An in vitro assay was used to determine the effects of conditioning nerve lesions on the regeneration of adult rat retinal ganglion cell (RGC) axons from retinal explants. Following the conditioning lesion (CL) of unilateral optic nerve transection, maximal regrowth was seen from RGC explanted from ipsilateral retinae 10 days post-CL. Explants from this group initiated axonal regrowth earlier and a greater percentage regrew axons when compared with explants from normal rats. Axonal regrowth from explants of retinae contralateral to CL was also seen earlier than normal. In further experiments, the effects of both exposure of the optic nerve sheath in the orbit and the incision of the dura without injury to optic nerve axons were studied. The conditioning effect of a dural incision was found to be the same as that of optic nerve transection, whilst exposure of the optic nerve sheath had no conditioning effect on RGC axonal regrowth in vitro.     

Book Review: Promoting Children's Health: Integrating School, Family, and Community, The Guilford Press (2003). Thomas Power, George DuPaul, Edward Shapiro, & Anne Kazak, 2003.

Pediatric, child clinical, and school psychologists will benefitfrom Promoting Children's Health, a 2003 publication by leadersin the field who have developed a practical text that is clearlybased upon a comprehensive review of theoretical models andresearch data. The work is likely to be embraced by a wide arrayof specialty psychologists. Further, Promoting Children's Healthhas the potential to unify distinctly specialized practice areas.In fact, this book will be in demand not only by practitionersin the field, but also by academic faculty who might add thisto reading lists for training graduate students. The authors     

Diagnosis of imported malaria with multiplex PCR on LighCycler apparatus

For the diagnosis of imported malaria, a competitive multiplex PCR using LightCycler was developed and compared for 3 months with a traditional PCR method. Hundred eighty three patients were tested by these 2 techniques of molecular diagnosis: 60 were positive for P. falciparum, 9 were positive for other Plasmodium species and 114 were negative. The LightCycler method was found in total agreement with the traditional PCR.     

Cytology and lineage of NG2-positive glia

We present evidence that NG2+ glia are an integral part of an oligodendrocyte/synantocyte (OS) lineage stream the progenitors of which begin to produce both glial phenotypes at about birth. The NG2 CSPG is differentially distributed within the OS lineage, being expressed in progenitors and synantocytes but not in oligodendrocytes. All cells in the OS lineage, except the primordial stem cells, express O4. The oligodendrocyte line reacts with CD9, but synantocytes are CD9–. Nonetheless, synantocytes are morphologically complex and specialised glia which contact axolemma in myelinated fibres at nodes of Ranvier and synaptic terminals, and form >99% of all NG2+ glia in the adult CNS. Thus, the other NG2+ phenotype, the adult oligodendrocyte progenitor cell (AOPC), constitutes a small population of <1% of all NG2+ glia in the mature CNS. AOPC are a heterogeneous set of cells probably originating from multiple sources which, by definition, produce oligodendrocytes in the adult to replace loss after trauma, demyelination and normal wear and tear. The definitive functions of synantocytes remain undefined.     

Three-dimensional culture of rat exocrine pancreatic cells using collagen gels

Rat pancreatic cells were dissociated using a combined enzyme and EDTA method, grown on a plastic surface and then overlayed with collagen gel. Our studies have shown that exocrine pancreatic cells grown in this way have the ability to rearrange themselves into a three-dimensional organoid structure in which well defined epithelial lumina have been identified by ultrastructural and light microscopic examination. This in vitro system has advantages in examining the cytodifferentiation of pancreatic cells and may be exploited in studying pancreatic carcinogenesis.