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291.
Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures
Zhou W Bibila T Glazomitsky K Montalyo J Chan C Distefano D Munshi S Robinson D Buckland B Aunins J 《Cytotechnology》1996,22(1-3):239-250
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. 相似文献
292.
The influence of mediterranean pine vole (Microtus (Terricola) duodecimcostatus) mound-building activity in two Western Spanish Pyrenees plant communities (Mesobromion erecti and Festucion eskiae-Nardion strictae) were studied. The plants colonizing the gaps in these areas are different in the two cases considered. The plant composition of surrounding plant communities seems to be the main factor in revegetation. Mound-building activity changes the species' relative frequency and life-form spectrum, decreases the monocytyledonous/dicotyledonous ratio and increases diversity by diminishing the presence of dominant plant species.Abbreviations ME
Mesobromion erecti
- FN
Festucion eskiae-Nardion strictae
- (monocots)
Monocotyledonous
- (dicots)
Dicotyledonous 相似文献
293.
Gerhard Bringmann Klaus-Peter Gulden Daniel Vitt Klaus Birken Clemens Helf 《Journal of molecular modeling》1995,1(3):161-175
AdaptivSearch is the first adaptive strategy based algorithm for the rational and economical construction of n-dimensional hypersurfaces. AdaptivSearch works iteratively: At each step it parcels out the definition range into several triangles, evaluates the worst according to a built-in error criterion, and refines the approximation to the unknown function by choosing the barycenter of this partial area as the node to be calculated next. Based upon the error criterion, AdaptivSearch selectively approaches those parts of the hypersurface in which the curvature exhibits the strongest changes. Some examples of AdaptivSearch applications for both analytical functions and chemical model surfaces are given in order to demonstrate the behavior of the algorithm. These show its broad applicability and the usefulness, especially for chemical problems. 相似文献
294.
BESSESEN, DANIEL H, CONNIE L RUPP AND ROBERT H ECKEL. Dietary fat is shunted away from oxidation, toward storage in obese zucker rats. Obes Res. 1995;3:179–189. Previous measurements of lipoprotein lipase (LPL) activity in adipose tissue (ATLPL) of lean and obese Zucker rats have consistently documented increased activity in obese rats relative to lean. Since LPL is considered to be rate limiting for the delivery of triglyceride fatty acids (TGFA) to muscle and adipose tissue, these data have been used to suggest that the metabolic partitioning of TGFA favors storage over oxidation in obese rats. To document the partitioning of TGFA directly, the fate of 14C labeled oleic acid (42nmols) was fed to lean, obese, and obese Zucker rats fed a hypocaloric diet designed to chronically reduce weight 25% below that of obese controls (reduced-obese). The amount of 14C recovered in CO2 over 6 hours following ingestion was significantly less in obese rats compared to lean (0.45 ± 0.06 vs. 0.88 ± 0.09nmols, p=.0004) and less still in the reduced obese group (0.34 ± 0.06nmols p=.00003). Six hours after ingestion, the quantity of label found in adipose tissue was significantly greater in the obese rats compared to lean (14.51 ± 1.92 vs. 1.38 ± 0.29nmols p<.00001), but was intermediate in the reduced-obese group (9.23 ± 0.98nmols p=.0003). At 2.2 hours there was significantly more label in skeletal muscle of lean rats compared to either obese or reduced-obese (2.33 ± 0.24; 1.35 ± 0.04nmols p=.01; 1.41 ± 0.27nm p=.02). However, at 6 hours these differences between groups were no longer present. These findings Indicate that dietary fat is shunted away from oxidation toward storage in obese Zucker rats. Additionally it appears that there may be a relative block in the oxidation of TGFA that is taken up by skeletal muscle in obese rats. Finally the relative normalization of this partitioning defect in reduced-obese rats is at variance with what was suggested by previous measurements of tissue specific levels of LPL, and suggests an enhanced recirculation of fatty acids from adipose tissue to muscle in reduced-obese rats. This could occur through increased delivery of non-esterified fatty acids (NEFA) to muscle as a result of an increase in net lipolysis. 相似文献
295.
Roberto E. Izquierdo Kimberly Breese Shalini Jain Daniel Carestio Lawrence Jung James Figge 《In vitro cellular & developmental biology. Animal》1995,31(1):71-76
Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH),
that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered
C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard,
a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture.
A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were
positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression
from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components
of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation
of transferred genes in animals. 相似文献
296.
297.
298.
Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers 总被引:9,自引:2,他引:7 下载免费PDF全文
Olivier Clermont Philippe Burlet Lydie Burglen Suzie Lefebvre Fabrice Pascal John McPherson John J. Wasmuth Daniel Cohen Denis Le Paslier Jean Weissenbach Mark Lathrop Arnold Munnich Judith Melki 《American journal of human genetics》1994,54(4):687-694
The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene. 相似文献
299.
Murielle Rinaldi-Carmona Francis Barth Michel Haulme David Shire Bernard Calandra Christian Congy Serge Martinez Jeanne Maruani Gervais Nliat Daniel Caput Pascual Ferrara Philippe Soubri Jean Claude Brelire Grard Le Fur 《FEBS letters》1994,350(2-3):240-244
SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system. 相似文献
300.
John P. Carulli De-Mao Chen William S. Stark Daniel L. Hartl 《Journal of molecular evolution》1994,38(3):250-262
Phylogenetic and physiological methods were used to study the evolution of the opsin gene family in Drosophila. A phylogeny based on DNA sequences from 13 opsin genes including representatives from the two major subgenera of Drosophila shows six major, well-supported clades: The blue opsin clade includes all of the Rhl and Rh2 genes and is separated into two distinct subclades of Rhl sequences and Rh2 sequences; the ultraviolet opsin clade includes all Rh3 and Rh4 genes and bifurcates into separate Rh3 and Rh4 clades. The duplications that generated this gene family most likely took place before the evolution of the subgenera Drosophila and Sophophora and their component species groups. Numerous changes have occurred in these genes since the duplications, including the loss and/or gain of introns in the different genes and even within the Rhl and Rh4 clades. Despite these changes, the spectral sensitivity of each of the opsins has remained remarkably fixed in a sample of four species representing two species groups in each of the two subgenera. All of the strains that were investigated had R1-6 (Rhl) spectral sensitivity curves that peaked at or near 480 nm, R7 (Rh3 and Rh4) peaks in the ultraviolet range, and ocellar (Rh2) peaks near 420 nm. Each of the four gene clades on the phylogeny exhibits very conservative patterns of amino acid replacement in domains of the protein thought to influence spectral sen sitivity, reflecting strong constraints on the spectrum of light visible to Drosophila. 相似文献