Tobacco use is the leading cause of preventable death in the United States. Four of every five persons who use tobacco begin before they reach adulthood; more than 3,000 young persons begin smoking each day. In addition, smoking is addictive-three of four teenagers who smoke have made at least one serious, yet unsuccessful, effort to quit. The importance of tobacco use cessation programs for youth is addressed in Healthy People 2000: National Health Promotion and Disease Prevention Objectives and in recently passed legislation related to the Goals 2000 National Education Goals. CDC's Guidelines for School Health Programs to Prevent Tobacco Use and Addiction states that tobacco cessation programs are needed to help young persons who already use tobacco. In 1994, both the Surgeon General's Report, Preventing Tobacco Use among Young People, and the Institute of Medicine's report, Growing Up Tobacco Free, indicated that there were very few effective cessation programs for youth and that more research is needed in this area. This project convened experts to provide recommendations on the design of a tobacco cessation intervention for youth, including helping pregnant teenagers who smoke to stop. This program is based on effective adult cessation programs with modifications relevant to adolescent development. During the first year the major foundational work for this project was accomplished. A database of key contacts and other related interventions in tobacco cessation for youth was developed, a review and analysis of prevalence and trends in adolescent smoking were conducted, and a cooperative agreement with the American Medical Association was established to complete the design, implementation, and evaluation of an effective tobacco cessation program for youth. During the second project year, this program was tested through quasi-experimental research at various school-based health clinics throughout the country. The third year involved follow-up data collection and program modification based on the results. Plans for dissemination of the intervention to youth-serving agencies across the nation will be developed. This is a 3-year project.

Adolescent↗

PubMedDate not supplied

293SF metabolic flux analysis during cell growth and infection with an adenoviral vector.

Metabolic flux quantification of cell culture is becoming a crucial means to improve cell growth as well as protein and vector productions. The technique allows rapid determination of cell culture status, thus providing a tool for further feeding improvements. Herein, we report on key results of a metabolic investigation using 293 cells adapted to suspension and serum-free medium (293SF) during growth and infection with an adenoviral vector encoding the green fluorescence protein (GFP). The model developed contains 35 fluxes, which include the main fluxes of glycolysis, glutaminolysis, and amino acids pathways. It requires specific consumption and production rate measurements of amino acids, glucose, lactate, NH(3), and O(2), as well as DNA and total proteins biosynthesis rate measurements. Also, it was found that extracellular protein concentration measurement is important for flux calculation accuracy. With this model, we are able to describe the 293SF cell metabolism, grown under different culture conditions in a 3-L controlled bioreactor for batch and fed-batch with low glucose. The metabolism is also investigated during infection under two different feeding strategies: a fed-batch starting at the end of the growth phase and extending during infection without medium change and a fed-batch after infection following medium renewal. Differences in metabolism are observed between growth and infection, as well as between the different feeding strategies, thus providing a better understanding of the general metabolism.

Adenoviridae↗

PubMedDate not supplied

Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism.

Growth, nutrient consumption, and end-product accumulation were quantitated in shake-flask cultures of two insect cell lines, Sf-9 and BTI-EAA, in three different serum-supplemented media. Per cell consumption or production rates were calculated for most medium components analyzed. Glucose was growth-limiting in TNM-FH medium and was the most important single source of organic-C for the cells in all cultures. Cells utilized fructose and maltose but not sucrose. alpha-Ketoglutarate and malate contributed significantly to the carbon budget of cells in TNM-FH. Lactate generally did not accumulate during growth. Most of the amino acids were consumed by the cells, with the exception of alanine which was produced. Most of the amino acids appeared to be present in adequate supply in the cultures. Glutamate was generally the most rapidly consumed of the amino acids, followed closely by glutamine. Alanine accumulation was correlated with glucose consumption. In Sf-9 cultures, ammonia accumulated only slightly or not at all as long as glucose was present in the medium, and uric acid was detectable at the end of growth and in the stationary phase. Added ammonia up to a concentration of 10 mM did not affect the growth of either cell line. Ammonia and lactate may be of less importance in limiting growth in insect cell cultures than in mammalian cell cultures. A hypothetical outline of the major metabolic pathways of the cultured insect cells is presented on the basis of information obtained here and in the literature.

Amino Acids↗

PubMedDate not supplied

Study of adenovirus production in serum-free 293SF suspension culture by GFP-expression monitoring.

The red-shifted S65T mutant green fluorescent protein (GFP) was used to compare the adenovirus (Ad) production and post-infection survival of 293SF and 293S cells in serum-free and serum-containing flask cultures, respectively. The GFP-expressing vector permitted the quantification of both the level of GFP expressed by infected cells and the infectious viral content of the cultures by flow cytometry in a simple, fast, sensitive, and reliable way. The GFP has the main advantage of fluorescing without any substrate addition. Infected cultures showed the coexistence of two populations of fluorescent cells, high-fluorescence cells (HFCs) and low-flourescence cells (LFCs), in proportions that varied between 20 and 75 hpi. The gradual increase in the number of LFCs at the expense of HFCs correlated well with the increase in the number of dead cells. This relationship could be used for the continuous measure of a culture's viability with the appropriate on-line instrumentation. The post-infection death rate of infected 293SF cells was higher than that of infected 293S cells, but the level of GFP fluorescence in viable, highly fluorescent cells was similar in the two infected cell lines. The number of infectious viral particles (IVPs) was quantified in less than 24 h by an infection assay of 293S cells in wells with viral particles extracted from the culture samples, and the results were more reproducible (+/- 10% variation) than those generally reported for conventional plaque assay titrations or end-point dilutions. The viable cell-specific IVP concentrations were for most experiments similar, indicating again that the difference between the two cell lines was their unequal post-infection viabilities, not the virus production by the infected living cells.

Adenoviridae↗

PubMedDate not supplied

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.

Ammonia↗

PubMedDate not supplied