Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.     

Dissimilarity-based algorithms for selecting structurally diverse sets of compounds.

This paper commences with a brief introduction to modern techniques for the computational analysis of molecular diversity and the design of combinatorial libraries. It then reviews dissimilarity-based algorithms for the selection of structurally diverse sets of compounds in chemical databases. Procedures are described for selecting a diverse subset of an entire database, and for selecting diverse combinatorial libraries using both reagent-based and product-based selection.     

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.     

Effect of training on the rating of perceived exertion at the ventilatory threshold

The purpose of this study was to determine the effect of training on the rating of perceived exertion (RPE) at the ventilatory threshold. College students were assigned to either training (n = 17) or control (n = 10) groups. Trainers completed 18 interval training sessions (five X 5 min cycling at 90-100% VO2max) and 8 continuous training sessions (40 min running or cycling) in 6 weeks. Pre- and post-training, cardiorespiratory, metabolic, and perceptual variables were measured at the ventilatory threshold during graded exercise tests on a cycle ergometer. Ventilatory threshold was that point above which VE X VO2-1 increased abruptly relative to work rate. Post-training means of trained and control subjects were compared using analysis of covariance, with pre-training values as covariates. Following training, the adjusted means for the trained subjects were significantly greater (p less than 0.05) than for controls for VO2max (6%), and for work rate (20%), VO2 (23%), and %VO2max (13%) at the ventilatory threshold. However, adjusted means for RPE at the ventilatory threshold were not significantly different (2%). Both before and after training, exercise at the ventilatory threshold was perceived as 'somewhat hard' to 'hard' (RPE = 13-15) by both groups. The relationship between RPE and %VO2max was altered by training, with trained subjects having a lower RPE at a given %VO2max. It is concluded that RPE at the ventilatory threshold is not affected by training, despite that after training the ventilatory threshold occurs at a higher work rate and is associated with higher absolute and relative metabolic and cardiorespiratory demands.     

Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells

Methylisocyanate (MIC) induced mutagenic responses in the absence of exogenous activation in the mouse lymphoma cell forward mutation assay at concentrations as low as 8-24 microM. MIC produced predominantly small mutant colonies, suggesting the possibility of clastogenic activity. The intermediate hydrolysis product, methylamine, was also mutagenic without exogenous activation but required several hundred-fold higher concentrations (ca. 3 mM). N,N'-Dimethylurea, the final product in the reaction of methylisocyanate and water, was totally refractory in either the presence or absence of S9 for concentrations up to 57 mM (5 mg/ml). The ethyl ester of N-methylcarbamic acid was also tested since it was the only available analogue to the highly reactive N-methylcarbamic acid intermediate. This compound was mutagenic only in the presence of S9 at doses exceeding 5-40 microM, which suggested the possibility that the free acid, produced by enzymatic hydrolysis, is also mutagenic. The mutagenic activity of the ester resulted solely in the production of small mutant colonies.     

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.     

Inhibitors of angiotensin-converting enzyme containing a tetrahedral arsenic atom.

A series of tetrahedral oxo acids of Group VA and VIA elements and of silicon and boron were examined as inhibitors of angiotensin-converting enzyme. Arsenate is a competitive inhibitor with a Ki of 27 +/- 1 mM, at least 10-fold more potent than phosphate. Dimethylarsinate is a competitive inhibitor with a Ki of 70 +/- 9 mM, 2-fold more potent than dimethylphosphinate. Oxo acids of boron, silicon, antimony, sulphur and selenium are not inhibitors. On the basis of these results and the strong inhibition of this zinc metallopeptidase by substrate analogues containing a tetrahedral phosphorus atom, two substrate analogues containing a tetrahedral arsenic atom were prepared. 2-Arsonoacetyl-L-proline is a competitive inhibitor with a Ki of 18 +/- 7 mM, more than 2000-fold weaker than that of its phosphorus analogue 2-phosphonoacetyl-L-proline. 4-Arsono-2-benzylbutanoic acid is a mixed inhibitor with a Ki of 0.5 +/- 0.2 mM, indistinguishable in potency from its phosphorus analogue 2-benzyl-4-phosphonobutanoic acid.     

Dependence on blood acetate concentration of the metabolic effects of ethanol in perfused rat liver

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5'-phosphate group react with nucleoside 5'- phosphorimidazolides in aqueous solution to give 'capped' pyrophosphates in at least 70% yield. If adenosine 5'- phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.