VIP gene transcription is regulated by far upstream enhancer and repressor elements.

SK-N-SH human neuroblastoma subclones differ widely in basal and second messenger induction of the gene encoding the neuropeptide vasoactive intestinal peptide (VIP). These differences were recapitulated by a chimeric gene which consisted of 5.2 kb of the human VIP gene 5' flanking sequence fused to a reporter. Subsequent gene deletion experiments revealed several regulatory regions on the gene, including a 645-bp sequence located approximately 4.0 upstream from the transcription start site. Here we examined this upstream region in detail. Inhibitory sequences were found to be present on each end of the 645-bp fragment. When removed, basal transcription increased more than 50-fold. Subsequent deletion/mutation analysis showed that the 213-bp fragment contained at least two enhancer elements. One of these was localized to an AT-rich 42-bp sequence shown by others to bind Oct proteins in neuroblastoma cells, while the other corresponded to a composite AP-1/ets element. In addition to these enhancers, a 28-bp sequence on the 213-bp fragment with no apparent homology to known silencers inhibited transcription. The studies provide molecular details of a complex regulatory region on the VIP gene that is likely to be used to finely tune the level of gene transcription in vivo.

Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects

A bacterial phytoene synthase (crtB) gene was overexpressed in a seed-specific manner and the protein product targeted to the plastid in Brassica napus (canola). The resultant embryos from these transgenic plants were visibly orange and the mature seed contained up to a 50-fold increase in carotenoids. The predominant carotenoids accumulating in the seeds of the transgenic plants were alpha and beta-carotene. Other precursors such as phytoene were also detected. Lutein, the predominant carotenoid in control seeds, was not substantially increased in the transgenics. The total amount of carotenoids in these seeds is now equivalent to or greater than those seen in the mesocarp of oil palm. Other metabolites in the isoprenoid pathway were examined in these seeds. Sterol levels remained essentially the same, while tocopherol levels decreased significantly as compared to non-transgenic controls. Chlorophyll levels were also reduced in developing transgenic seed. Additionally, the fatty acyl composition was altered with the transgenic seeds having a relatively higher percentage of the 18 : 1 (oleic acid) component and a decreased percentage of the 18 : 2 (linoleic acid) and 18 : 3 (linolenic acid) components. This dramatic increase in flux through the carotenoid pathway and the other metabolic effects are discussed.

Poly(beta-hydroxybutyrate) production in oilseed leukoplasts of brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(beta-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760-12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a beta-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(beta-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation.

Targeting of embryonic and postnatal autonomic and enteric neurons with a vasoactive intestinal peptide transgene.

The neuropeptide vasoactive intestinal peptide (VIP) is expressed in several distinct sites in the CNS, in cholinergic and enteric ganglia, and in a small subpopulation of neurons within sympathetic ganglia. Previous studies on the human VIP gene indicate that transcription in neural crest-derived neuroblastoma and pheochromocytoma cell lines is controlled in part by multiple regulatory elements located along 4.5 kb of upstream 5' flanking sequence. In the current studies, transgenic mice were created with a chimeric gene consisting of 16.5 kb of the mouse VIP gene fused to the beta-galactosidase reporter. In situ hybridization analysis in adult mice indicated that reporter gene expression was correctly targeted to neurons in the esophagus, stomach, small intestine, and colon. No expression was observed in the brain, including regions that contain abundant VIP-expressing cells, such as the thalamus, amygdala, cerebral cortex, hippocampus, and suprachiasmatic nucleus. Analysis of transgene expression in neonatal and embryonic day 13.5 mice revealed a near perfect correlation between VIP and beta-galactosidase gene expression in cranial cholinergic ganglia and the superior cervical ganglia, and lack of transgene expression in sensory ganglia and in nonneuronal tissue. Potential ectopic transgene expression was observed in neonates, in the cerebellar external granule layer and in a small subpopulation of neurons in the olfactory epithelium. We conclude that the 16.5 kb of VIP gene used in these studies contains sequences sufficient for directing expression specifically to VIP neurons in the PNS, and that sequences located elsewhere on the gene are required for proper CNS expression. The VIP gene sequences used here should be capable of targeting other gene products to specific populations of embryonic and adult peripheral neurons without causing significant expression in the CNS.

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: a potential bioartificial liver.

Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.

A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids.

A simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi bodies, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.

Distinct regulation of vasoactive intestinal peptide (VIP) expression at mRNA and peptide levels in human neuroblastoma cells.

Neuronal differentiation was induced in cultures of the human neuroblastoma cell line subclone SH-SY5Y by 14-day treatment with dibutyryl cAMP (dBcAMP), retinoic acid, and phorbol 12-myristate 13-acetate (PMA). An approximate 4-fold increase in vasoactive intestinal peptide (VIP) mRNA concentration was observed after differentiation with retinoic acid, whereas no change in VIP mRNA concentration was observed after differentiation with dBcAMP or PMA. A short-term treatment of cells with PMA did however result in a 5-fold transient increase in VIP mRNA; prior differentiation with retinoic acid or dBcAMP diminished this effect. Observed increases in VIP mRNA were in all cases accompanied by increases in VIP immunoreactivity. Remarkably, however, long-term treatment of cells with dBcAMP, which caused no change in mRNA levels, resulted in a six-fold increase in VIP immunoreactivity. Acute (36-h) treatment with carbachol also caused an increase in VIP immunoreactivity (about 2-fold, and blocked by atropine) without an increase in VIP mRNA level. Thus, a quantitative change in gene transcription or mRNA stability appears not to be a prerequisite for increased VIP expression, indicating that regulation can occur at translational or post-translational steps.

Human alpha tumor necrosis factor does not damage cultures containing rat Schwann cells and sensory neurons.

The effects of recombinant human alpha tumor necrosis factor (alpha-TNF) were compared with those of cytotoxic serum from patients with the acute Guillain-Barré syndrome (GBS) in myelinated cultures containing only rat Schwann cells and dorsal root ganglion neurons. Alpha-TNF did not damage rat peripheral nervous system tissue in culture. These observations suggest that alpha-TNF is not responsible for the cytotoxic activity of acute GBS serum in culture.

Maturation of soybean somatic embryos and the transition to plantlet growth.

The maturation of soybean (Glycine max L. Merr.) somatic embryos was characterized. Maturation was assayed by evaluating the ability of somatic embryos to make the transition to a plantlet through a germination-like process. Somatic embryos were organized from cotyledons of immature soybean embryos. Maturation of somatic embryos occurred on a Murashige-Skoog basal medium supplemented with activated charcoal and 0.28 molar sucrose. After 8 weeks on this medium, somatic embryos exhibited vigorous, high frequency development to plantlets. The "germination" frequency (conversion) of somatic embryos, and plantlet recovery frequency varied concurrently with maturation period. Conversion and plant recovery required no exogenous growth regulators. Desiccation of immature somatic embryos under controlled humidity regimes resulted in increased frequency of conversion of immature somatic embryos. Morphological abnormalities appeared in the somatic embryos, but few were detrimental to conversion velocity. There was little effect of genotype on conversion velocity or frequency.

Sound localization in subjects with impaired hearing. Spatial-discrimination and interaural-discrimination tests.

In order to get a systematic picture of how various hearing impairments and neurologic disorders may affect sound localization, psychophysical spatial- and lateralization-discrimination measurements were performed on 140 subjects, including 69 with different types of hearing impairments, 32 with neurological diseases, and 39 with normal hearing. The quantities measured were: in the freefield, the horizontal minimum audible angle (MAA) at eight reference azimuths around the head and the vertical MAA straight ahead; with headphones, the just-noticeable difference (JND) in interaural time delay and the JND in interaural intensity difference. The standard stimulus was broadband (0.25-10 kHz), pulsed (1-sec), noise presented at a suprathreshold level for both ears (65-100 dB SPL). The results show that there exist characteristic impairments of sound localization in the different types of hearing impairments tested. On a general level, the results are consistent with the concept that the localization of sound relies on a decision made by the central auditory system based on a number of cues present in the acoustic signal at the two ears. The cues tested in our study are: 1) the interaural time difference, 2) the interaural intensity difference, and 3) the spectrum of the received signal at each ear. At a more specific level, the sound localization impairments found in conductive hearing losses are interpreted as bone-conduction effects, the results found in sensorineural hearing losses are interpreted as consequences of impaired or preserved spectral processing, the results in neurinomas are interpreted as impaired signal transmission in the auditory nerves, and the results of subjects with central involvements suggest that separate processors exist at some level in the central auditory system for the different localization cues. Finally, comments are made about the practical clinical significance of sound localization tests in the audiological and neurological evaluation of patients.