High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties

Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.     

Exploration of the two‐photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser

We have studied the wavelength dependence of the two‐photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin‐ and cellulose‐staining dye Calcofluor White and Alexa Fluor 350, in the visible and near‐infrared wavelength range (540–800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two‐photon microscopy. We also find that although the rate of photo‐bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution‐based measurements.     

Dietary intake of Senegalese adults

The aim of this work is to identify major food sources and dietary constituents of Senegalese adults. We conducted a cross-sectional study, using a single 24-hour dietary recall interview. Foods were classified into food groups based on similarities in nutrient content or use. Food groups included foods consumed individually, or as part of food mixtures such as stews, soups, or sandwiches. Median consumption (amount/day) of each food was determined and examined by relevant subgroups. Participants were 50 healthy Senegalese men, aged 20-62 years recruited at the Hôpital Général de Grand Yoff in Dakar, Senegal and from Sendou village, a rural area outside Dakar. A total of 90 foods and beverages were identified and classified into 11 groups. Sixty-five percent of foods identified could be classified as meats, grains, or fruits/vegetables. Fruits and vegetables comprised 42% (38/90) of all foods; meats 12% (11/90); and grains 11% (10/90). Sauces (6%, 5/90), sweets (4%, 4/90), and desserts (4%, 4/90) were also reported. The most common fruits/vegetables reported were potato, carrot, mango, and lettuce; commonly reported grains were bread and rice; and commonly reported meats were fish, beef, and ox. There were no differences in reported daily intake of each food by age, ethnicity, education, or residence. Most foods reported were traditional to the Senegalese diet, despite the increasing availability of Western foods in Senegal.     

The role of state policies and programs in buffering the effects of poverty on children's immunization receipt

OBJECTIVES: This study assessed the influence of public policies on the immunization status of 2-year old children in the United States. METHODS: Up-to-dateness for the primary immunization series was assessed in a national sample of 8100 children from the 1988 National Maternal and Infant Health Survey and its 1991 Longitudinal Follow-Up. RESULTS: Documented immunization rates of this sample were 33% for poor children and 44% for others. More widespread Medicated coverage was associated with greater likelihood of up-to-dateness among poor children. Up-to-dateness was more likely for poor children with public rather than private sources of routine pediatric care, but all children living in states where most immunizations were delivered in the public sector were less likely to be up to date. Poor children in state with partial vaccine replacement programs were less likely to be up to date than those in free-market purchase states. CONCLUSIONS: While state policies can enhance immunization delivery for poor children, heavy reliance on public sector immunization does not ensure timely receipt of vaccines. Public- and private-sector collaboration is necessary to protect children from vaccine-preventable diseases.