S-alk(en)yl-L-cysteine sulfoxides, alliinase and aroma in Leucocoryne.

Levels of S-alk(en)yl-L-cysteine sulfoxides, alliinase and enzymatically generated pyruvic acid were determined in the bulb, leaf and scape of five species and a natural hybrid of Leucocoryne (Liliaceae), a genus of ornamental geophytes indigenous to Chile. (+)-S-Methyl-L-cysteine sulfoxide (MCSO) was present in all plant parts of all species at levels between 0.09 and 1.41 mg g(-1) fr. wt. Trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (PRENCSO) was present in plant parts of three species only (L. angustipetala, L. oadorata and L. purpurea) at levels between 0.12 and 1.82 mg g(-1) fr. wt. No other S-alk(en)yl-L-cysteine sulfoxides were detected. Alliinase (EC 4.4.1.4) was detected in the leaf, bulb and scape of L. angustipetala and L. purpurea, only in the leaves of L. coquimbensis and L. purpurea x L. coquimbensis, and only in the bulb of L. odorata. Enzymatically generated pyruvic acid was detected in all plant parts of all species at levels between trace amounts and 5.33 micromol g(-1) fr. wt. As PRENCSO is produced only in Leucocoryne species exhibiting a strong and unpleasant onion-like aroma, it is probable that the enzymatic degradation of PRENCSO is the main cause of that aroma. Consequently, Leucocoryne cultivars should be selected in species and hybrids that lack the ability to synthesise PRENCSO.

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe.

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid.

The secretion of active recombinant human gastric lipase by Saccharomyces cerevisiae.

The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.

Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe.

A cDNA encoding human gastric lipase (hGL) has been expressed on multicopy plasmids in the fission yeast Schizosaccharomyces pombe (Sp). Active lipase is secreted from transformants containing the hGL cDNA under the control of either the Sp adh1 promoter (Padh1) or the plant cauliflower mosaic virus (CaMV) 35S promoter. Cell-wall-associated lipase activities are greatest in the early logarithmic growth phase and with Padh1. Western blot analysis indicates that a protein of identical molecular mass to natural hGL is secreted by Sp, although the major secreted product is of a higher molecular mass than either native hGL or recombinant hGL produced in the budding yeast Saccharomyces cerevisiae (Sc). Several distinct hGL are present within cells at all growth phases. Treatment of these proteins with endoglycosidase H gives rise to a single species equivalent in size to deglycosylated natural hGL, indicating that most of these are glycosylation intermediates. An hGL of similar molecular mass accumulates intracellularly in Sp when a modified version of cDNA is used which lacks the sequence encoding the natural secretory signal peptide. Production of hGL markedly slows the growth rate of Sp. The average copy number per cell of the plasmid expressing the hGL cDNA from the recombinant Padh1 is 2-3, as compared with 11-12 for the control plasmid.

Effect of Hydrogen Cyanamide on Amino Acid Profiles in Kiwifruit Buds during Budbreak.

Buds, and resultant shoots, were collected from kiwifruit (Actinidia deliciosa [A. Chev.] CF Liang et AR Ferguson var deliciosa cv Hayward) vines from late autumn until late spring with and without hydrogen cyanamide treatment. Those samples were weighed and analyzed for total nitrogen and free amino acids. By 7 days after hydrogen cyanamide treatment, the amount of proline had risen to nearly one-quarter of the total amino acid pool in the treated buds and that proportion was maintained for at least 14 days before it declined. The maximum concentration detected in treated buds was 25.8 micromoles per gram dry weight, 6.6 times that detected in untreated buds. By 95 days after treatment, the relative amounts of proline were not significantly different.

Carbohydrate requirements of peach fruit growth and respiration.

Data on the seasonal patterns of fruit growth and dark respiration of two peach (Prunus persica (L.) Batsch) cultivars were combined with temperature data to calculate the carbohydrate requirements of an "average" peach fruit from bloom to harvest. The two peach cultivars used were June Lady (an early maturing (mid-June) cultivar) and O'Henry (a late maturing (early-August) cultivar). At harvest, the mean dry weight of the June Lady fruit was 17.8 g (139.7 g fresh weight) and of O'Henry fruits was 30.9 g (213.9 g fresh weight), and the times from full bloom to harvest were 107 and 154 days, respectively. The total calculated fruit respiration requirements were 132 and 300 mmol CO(2) fruit(-1) season(-1) for June Lady and O'Henry fruits, respectively. Total calculated carbohydrate requirements for fruit growth and respiration are 23.9 and 43.8 g CH(2)O fruit(-1) season(-1) for June Lady and O'Henry fruits, respectively. Fruit respiration accounted for 16.3% of the total carbohydrate requirements of June Lady fruits and 0.5% of the total carbohydrate requirements of O'Henry fruits.

Effect of growth temperature upon heat sensitivity in Saccharomyces cerevisiae.

The resistance of exponentially growing yeast cells to killing by exposure to 52 degrees C increase markedly as the growth temperature was increased. Identical killing curves were obtained for cells suspended in growth medium or in 0.9% saline. Cells resistant to killing at 52 degrees C were quite sensitive to killing at slightly higher temperatures. These results suggest a primary role for membrane damage in the mechanism of heat killing.