Applications of BOP reagent in solid phase synthesis. Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1-29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa-fluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219-1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22-29) and (13-29) fragments of [Ala15]-GRF(1-29)-NH2. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala15]-GRF(1-29)-NH2 was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1-29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (less than 10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

Use of ars18 based vectors to increase protein production in Yarrowia lipolytica.

The isolation of ars sequence from the yeast Yarrowia lipolytica has recently been reported (Fournier et al., 1991). Vectors containing ars18 have been used to increase homologous and heterologous protein production. Examples presented are the Yarrowia lipolytica alkaline extracellular protease (AEP), the porcine alpha 1-interferon and the bovine prochymosin. A 2- to 6-fold increase in the corresponding protein production was observed and in several cases it was established that it corresponded to the copy number of plasmid in the cell.     

High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis.

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.     

Acetylcholinesterase from Drosophila melanogaster. Identification of two subunits encoded by the same gene

Purified acetylcholinesterase from Drosophila melanogaster is composed of a 55 kDa and a 16 kDa noncovalently associated subunit. Cleavage of disulfide bonds reveals that two 55 kDa polypeptides are linked together in native dimeric AChE. Western blots with two antibodies directed against the N- and C-termini of the predicted AChE primary sequence show that the 55 and 16 kDa polypeptides originate from proteolysis of the same precursor encoded by the Ace locus.     

A numerical evaluation of a hollow fiber extractive fermentor process for the production of ethanol

The economics of a process for the production of ethanol employing a hollow fiber extractive fermentor have been investigated. A computer simulation of the process incorporating a mathematical model of the fermentor was used to calculate the mass and energy balances. The results of the process simulation were read into a computer spreadsheet programmed with the economic calculations from which a final ethanol product cost was obtained. The process was found to be as competitive as conventional fermentation processes even at the currently high cost--$4/sq ft--of hollow fibers. It was determined that the 1986 price of 46.2 cents/L of ethanol produced by the process would be reduced by 1.8 cents/L for every $1/sq foot drop in the price of hollow fibers. A comparison of this process with conventional fermentation processes indicates that its potential savings lie in its ability to use a concentrated sugar feed, and the fermentor's increased productivity and ability to produce a concentrated ethanol stream which is removed by the extracting solvent.     

Mathematical model of microporous hollow-fiber membrane extractive fermentor

A mathematical model is presented for a microporous hollow-fiber membrane extractive fermentor (HFEF). The model is based on the continuous flow of the aqueous nutrient phase and cells through the shell space of the fermentor where the fermentation reaction occurs. The product diffuses from the shell space through the hollow-fiber membrane where it is continuously removed by solvent flowing concurrently through the fiber lumen. Results for ethanol production show that the HFEF has a volumetric productivity significantly higher than that possible using conventional methods. The model predicts the existence of an optimum volume fraction of hollow fibers in the fermentor that maximizes the total volumetric productivity. This optimum is the result of a classic trade-off between the volume fraction of the fermentor required for fermentation and that required for efficient removal of the ethanol product to minimize product inhibition.     

Sélection artificielle pour la résistance au méthidathion chezPhytoseiulus persimilis A. H.

Résumé La toxicité du méthidathion a été mesurée sur 7 souches dePhytoseiulus persimilis provenant de collections de laboratoire, d'unités de production ou du terrain. Les résultats obtenus se sont avérés être très variables: la CL50 de la souche la plus résistante est 100 fois plus élevée que celle de la souche la plus sensible. Chaque souche a ensuite été sélectionnée pour la résistance à cet insecticide; toutes les souches ont répondu à la sélection et ont atteint des niveaux de résistance similaires. Elles ont ensuite été mélangées, à nouveau sélectionnées pour la résistance au méthidathion et à d'autres insecticides et utilisées dans les programmes de lutte intégrée. La résistance a pu ainsi être légèrement augmentée et la souche obtenue, bien qu'encore affectée par le méthidathion sur le terrain, n'est plus complètement éliminée par les traitements et est moins sensible aux résidus présents sur les feuilles.      

Morphological aspect and chemical composition of Skeletonema costatum (Bacillariophyceae) growing in natural nutritive media with the aid of a culture system of dialysing fibers

The growth of Skeletonema costatum, under natural nutriment conditions, was studied using a bulk culture fiber dialyzing apparatus. The diatom displayed normal development of chain length (average cell number per chain) which coincided with the culture growth stages; that is, the cell number per colony increased during the active division period and decreased thereafter with the beginning of the prestationary phase. This morphological behaviour showed that the alga cells were not affected by such physical shocks as collision or tension occurring during repeated cell transfers from growth chambers to the dialyzing apparatus or at the time of their passage through the fiber fascicles. Measured at different growth stages, the cell contents in carbon, nitrogen, and chlorophyll confirmed the above results and showed for S. costatum a biological productivity comparable with that obtained in smaller dialyzing containers (dialyzing bags). Through a comparison between the dialyzing culture and a static culture grown in an enriched medium, certain characteristics were determined.     

Activité migratoire des tétranyques: Mise en évidence d'un rythme

Résumé L'étude en différentes conditions de la migration verticale deTetranychus urticae dans de fortes infestations, a permis de mettre en éviduence un rythme circadien caractérisé par un mouvement alterné de montée et de descente, la population migrant vers le haut des plantes en début d'après-midi. Ce rythme est en partie endogène puisqu'il se maintient plusieurs jours dans des conditions constantes.