The P2 element of the td intron is dispensable despite its normal role in splicing.

The P2 region of group I introns has been proposed to be involved in the correct positioning of the P1 5'-splice site duplex in the catalytic core (Michel, F., and Westhof, E. (1990) J. Mol. Biol. 216, 585-610). The behavior of delta P2 deletion mutants of the td intron is consistent with this hypothesis. The delta P2 mutants are capable of site-specific hydrolysis, indicating that the conformation of the ribozyme is not grossly altered, but they are incapable of transesterification reactions at the splice sites, as would be predicted if P1 is not appropriately aligned within the catalytic core. Nevertheless, the function of the P2 element can be bypassed in specific pseudorevertants isolated by genetic selection from the delta P2 mutants. These results, together with phylogenetic data, support the existence of alternate strategies to create a functional P1-core interaction.     

Feedback inhibition of polyisoprenyl pyrophosphate synthesis from mevalonate in vitro. Implications for protein prenylation.

The prenylation of proteins utilizes the polyisoprenyl pyrophosphates (FPP) and geranylgeranyl pyrophosphate (GGPP) as prenyl donors. These polyisoprenoids are also precursors to ubiquinone and dolichol synthesis. We have previously described the geranylgeranylation of rab 1b from labeled mevalonate in rabbit reticulocyte lysates (Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. C., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6264-6268). We now directly demonstrate the incorporation of mevalonate into FPP and GGPP in rabbit reticulocyte cytosol. High pressure liquid chromatography analysis reveals that only all-trans-E,E,E-GGPP, the prenyl donor for in vivo protein geranylgeranylation, is synthesized. Incubations with recombinant H-ras and rab1b result in an increased synthesis of farnesyl and geranylgeranyl derivatives, respectively. The increase is wholly accounted for by protein-incorporated polyisoprenoids with no change in the polyisoprenyl pyrophosphate pools. Further, GGPP inhibits its own synthesis, without affecting FPP synthesis, with half-maximal inhibition at approximately 3 microM GGPP. Inhibition of FPP synthesis by the inhibition of isopentenyl isomerase causes a dramatic increase in isopentenyl pyrophosphate synthesis. FPP also inhibits conversion of mevalonate into FPP. These findings indicate that these polyisoprenyl pyrophosphates can down-regulate their own synthesis in vitro, and this regulation may control the levels of these polyisoprenoids in vivo.     

Basement membrane lectin binding sites are decreased in the esophageal endoderm during the arrival of presumptive muscle mesenchyme in the developing asteroid Pisaster ochraceus.

Basement membranes (BMs) of vertebrates and invertebrates have been shown to contain glycoproteins and proteoglycans, which include oligosaccharides and glycosaminoglycans. Lectin binding sites were characterized in the BM of gastrulating embryos of the starfish, Pisaster ochraceus. In early and mid-gastrulae, the fluorescein isothiocyanate (FITC)-lectin conjugates of concanavalin A (Con A) and wheat germ agglutinin (WGA) reveal the presence of mannose/glucose and glucosamine/sialic acid residues in the BM of all regions of the embryos. However, in the late gastrula embryo, an apparent reduction of these components is observed over the esophageal BM. Ultrastructural studies using the lectin-gold conjugates Con A, Limax flavus agglutinin (LFA), specific for sialic acid, and Dolichos biflorus agglutinin (DBA), specific for galactosamine, demonstrate that most mannose/glucose and galactosamine-containing residues lie in the lamina densa, whereas most sialic acid residues are located over the lamina lucida. In addition, a statistical analysis of lectin binding in the late gastrula embryo reveals that the amount of labelling with both Con A and LFA is significantly reduced in the esophageal region, suggesting that mannose/glucose and sialic acid residues are reduced in this region. These results confirm the observations of the FITC-lectin studies described above. They also confirm earlier studies that demonstrated a difference in BM morphology of the esophageal region (Crawford, '88). Mesenchyme cells, some of which arise from the forming coeloms (Crawford, '90), and which may represent a distinct population, colonize exclusively on this esophageal BM, where they later differentiate into muscle. Quantitative differences in BM glycoconjugates may act to direct the presumptive muscle cells to the region of the esophagus.     

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant

Summary Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mm lactate exhibited intracellular lactate and glutamine levels of 40 mm and 4 mm, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mm lactate concentrations. For cells cultivated in medium supplemented with 6 mm pyruvate, intracellular lactate levels were estimated at approx. 40 mm, but these cells also showed an increased intracellular alanine concentration of 22 mm. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.     

Short chain flavour esters synthesis by microbial lipases

Summary The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.