The molecular mechanisms controlling the process of myelination by Schwann cells remain elusive, despite recent progress in the identification and characterization of genes encoding myelin components (reviewed in ref. 1). We have created a null allele in the mouse Krox-20 gene, which encodes a zinc-finger transcription factor, by in-frame insertion of the Escherichia coli lacZ gene, and have shown that hindbrain segmentation is affected in Krox-20-/- embryos. We demonstrate here that Krox-20 is also activated in Schwann cells before the onset of myelination and that its disruption blocks Schwann cells at an early stage in their differentiation, thus preventing myelination in the peripheral nervous system. In Krox-20-/- mice, Schwann cells wrap their cytoplasmic processes only one and a half turns around the axon, and although they express the early myelin marker, myelin-associated glycoprotein, late myelin gene products are absent, including those for protein zero and myelin basic protein. Therefore Krox-20 is likely to control a set of genes required for completion of myelination in the peripheral nervous system.

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calfserum for 2-12 days in vtitro (DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and ofnewly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K+ concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K+ concentration was small and Ca 2 independent, but it progressively and steadily increased (up to 8-to 10-fold) and became Ca2" dependent (up to 80-85%) at later stages (4, 8, and 12 DIV).Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. y-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that ofglutamate, and y-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K+ concentration-evoked release of this amino acid was modest and scarcely affected by Ca2". Neither high K+ concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.It is known that the excitatory information reaching the cerebellum through the mossy fibers is conveyed to the inhibitory neurons of the cerebellar cortex (mainly to the Purkinje cells) by excitatory interneurones-the granule cells (1), which represent the most abundant neuronal population of the cerebellar cortex. Although glutamic acid has been proposed as the excitatory transmitter of granule cells (2-7), largely on the basis of indirect neurochemical evidence, definitive proof substantiating this hypothesis is still lacking, and some data ofthe literature even appear to be in contrast with it. For example, the high affinity uptake and the concentration of glutamate were not found to be significantly decreased in cerebella severely depleted of granule cells (refs. 5 and 8; however, see refs. 2-7), as one would expect if a substantial glutamate pool were associated with these cells. Moreover, autoradiographic studies on cerebellar slices preincubated in the presence of low concentrati...

Generally, cationic vector-based intravenous delivery of at a ratio of 4 nitrogen equivalents per DNA phosphate. DNA is hindered by interactions of positively charged comLower levels of transfection were found in the heart, plexes with serum proteins. However, if optimally formuspleen, liver and kidney. Expression was dose-and timelated, cationic vectors can provide reasonable levels of dependent in all tissues examined. In the lung, ␤-galactotransfection in the lung either by intravenous or intrapulsidase staining showed transgene expression in clusters monary routes. We investigated the in vivo transfection of 10 or more pulmonary cells including the alveolar endocapacity of a cationic polymer: linear, 22 kDa polyethylenithelium, squamous and great alveolar epithelial cells (type mine. PEI/DNA complexes were formulated in 5% glucose I and II pneumocytes) and septal cells. These findings indiand delivered into adult mice through the tail vein. Two cate that the complexes pass the capillary barrier in the marker genes were used, ␤-galactosidase and luciferase.lung. Although the delivery mechanism requires eluciHigh levels of luciferase expression (10 7 RLU/mg protein) dation, linear PEI has promise as a vector for intravenous were found in the lung when DNA was complexed with PEI transfer of therapeutic genes.Keywords: cationic polymers; pneumocytes; plasmid DNA; nonviral; gene therapy A number of generations of cationic vectors have been synthetised and tested in a variety of in vivo models and some have been taken to clinical trials. Most of these vectors are either monocationic or polycationic lipids, but there has been more recent interest in cationic polymers. Indeed, we showed that the branched cationic polymer polyethylenimine (PEI) can provide high levels of transfection in vivo. [1][2][3] In particular the lowest molecular weight preparation then commercially available, the 25 kDa preparation from Aldrich, was shown to be a versatile and efficient vector in the mammalian brain. 2 In a more recent study, 3 we chose to examine the effect of formulation procedures (glucose or saline solutions) on the size and in vivo transfection activity of a mixture of linear polymers with a mean MW of 22 kDa (Exgene 500; Euromedex, Souffleweyersheim, France). We found that the complexes formed in glucose were an order of magnitude smaller than those formed in saline and these complexes provided high levels of gene transfer following dilution into a physiological medium, the cerebrospinal fluid. In the light of these findings we chose to examine the effects of injecting complexes of plasmid DNA formulated with 22 kDa PEI in 5% glucose directly into the blood system and to examine transgene expression in a variety of organs.PEI-DNA complexes with different ratios of PEI nitro- gen to DNA phosphate (N/P ratio) were prepared in 5% glucose using a CMV-Luc plasmid 4 and the 22 kDa linear PEI. This PEI is synthesised to a degree of polymerisation of 510 units. Earlier experiments carried out with the branched 25 kDa PEI (Aldri...