The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

Reductive dechlorination of high concentrations of tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis.

Tetrachloroethene, also known as perchloroethylene (PCE), is a common groundwater contaminant throughout the United States. The incomplete reductive dechlorination of PCE--resulting in accumulations of trichloroethene, dichloroethene isomers, and/or vinyl chloride--has been observed by many investigators in a wide variety of methanogenic environments. Previous mixed-culture studies have demonstrated that complete dechlorination to ethene is possible, although the final dechlorination step from vinyl chloride to ethene is rate limiting, with significant levels of vinyl chloride typically persisting. In this study, anaerobic methanol-PCE enrichment cultures which proved capable of dechlorinating high concentrations PCE to ethene were developed. Added concentrations of PCE as high as 550 microM (91-mg/liter nominal concentration; approximately 55-mg/liter actual aqueous concentration) were routinely dechlorinated to 80% ethene and 20% vinyl chloride within 2 days at 35 degrees C. The methanol level used was approximately twice that needed for complete dechlorination of PCE to ethene. The observed transformations occurred in the absence of methanogenesis, which was apparently inhibited by the high concentrations of PCE. When incubation was allowed to proceed for as long as 4 days, virtually complete conversion of PCE to ethene resulted, with less than 1% persisting as vinyl chloride. An electron balance demonstrated that methanol consumption was completely accounted for by dechlorination (31%) and acetate production (69%). The high volumetric rates of PCE dechlorination (up to 275 mumol/liter/day) and the relatively large fraction (ca. one-third) of the supplied electron donor used for dechlorination suggest that reductive dechlorination could be exploited for bioremediation of PCE-contaminated sites.     

Down''s Syndrome Individuals Begin Life with Normal Levels of Brain Cholinergic Markers

We measured the activities of the cholinergic marker enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in autopsied brains of seven infants (age range 3 months to 1 year) with Down's syndrome (DS), a disorder in which virtually all individuals will develop by middle age the neuropathological changes of Alzheimer's disease accompanied by a marked brain cholinergic reduction. When compared with age-matched controls cholinergic enzyme activity was normal in all brain regions of the individuals with infant DS with the exception of above-normal activity in the putamen (ChAT) and the occipital cortex (AChE). Our neurochemical observations suggest that DS individuals begin life with a normal complement of brain cholinergic neurons. This opens the possibility of early therapeutic intervention to prevent the development of brain cholinergic changes in patients with DS.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.