Production of heterologous polygalacturonase I from Aspergillus kawachii in Saccharomyces cerevisiae in batch and fed-batch cultures.

The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase.

Nicotine dependence among adolescent smokers.

OBJECTIVE: To assess nicotine dependence among adolescents to determine whether quitting smoking is associated with the emergence of nicotine withdrawal symptoms and craving, and to identify the factors associated with these symptoms. DESIGN: Cross-sectional survey. PARTICIPANTS: We studied 2197 10th-grade students in 6 San Jose, Calif, high schools. MAIN OUTCOME MEASURES: Smoking status; history of quitting smoking; Modified Fagerstrom Tolerance Questionnaire (mFTQ) scores; subjective nicotine withdrawal symptoms from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; Center for Epidemiologic Studies Depression Scale (CES-D); and saliva cotinine levels. RESULTS: Of the 485 participants who reported having smoked during the past 30 days, 249 reported previous attempts to quit smoking. Among the participants who had attempted to quit, the self-reported frequencies of subjective withdrawal symptoms were a strong need to smoke (45.4%), nervous and tense (31.8%), restless (29.4%), irritable (28.7%), hungry (25.3%), unable to concentrate (21.7%), miserable and sad (15.3%), and trouble sleeping (12.8%). The total number of withdrawal symptoms was correlated with the mFTQ score (Spearman r = 0.51; P < .001). In a stepwise linear regression analysis, the mFTQ score and the CES-D score accounted for approximately 35% of the variance in total number of withdrawal symptoms (R2 = 0.35; P < .001). Males smoked significantly more and had significantly higher mFTQ scores than did females, while female smokers had significantly higher CES-D scores than did their male counterparts. CONCLUSIONS: Considerable levels of nicotine dependence were present among adolescent smokers. Use of mFTQ scores; withdrawal symptoms including nicotine craving; CES-D scores; and saliva cotinine levels may be helpful in designing cessation programs targeted to nicotine-dependent adolescents.