Photoaffinity labeling of the human erythrocyte nucleoside transporter by N6-(p-Azidobenzyl)adenosine and nitrobenzylthioinosine. Evidence that the transporter is a band 4.5 polypeptide

N6-(p-Azidobenzyl)adenosine (ABA) and nitrobenzylthioinosine (NBMPR) were employed as covalent probes of the nucleoside transport mechanism in human erythrocytes. NBMPR, a potent inhibitor of nucleoside transport, binds tightly (KD 0.3-1 nM) to specific sites on nucleoside transporter elements. ABA, a less potent inhibitor of uridine influx, competitively inhibited NBMPR binding (Ki 15 nM). [3H]ABA was bound tightly (KD 13.4 nM) but reversibly to sites on erythrocytes which appeared to be those which bind NBMPR. ABA binding was inhibited by uridine and adenosine. Irradiation with UV light caused site-bound [3H]ABA on erythrocyte membranes to become covalently bound and, similarly, photoactivation resulted in covalent attachment of membrane-bound [3H]NBMPR. In the presence of dithiothreitol, a free radical scavenger, photoactivation of the site-bound 3H-ligand on membranes depleted of extrinsic membrane proteins resulted in selective incorporation of 3H into band 4.5 of the membrane polypeptides which were resolved on sodium dodecyl sulfate-polyacrylamide gel electropherograms. This result, when considered with previous findings, indicates that the NBMPR-binding component of the nucleoside transport mechanism (or the entire mechanism, if the NBMPR site is an integral part) is a band 4.5 polypeptide.     

Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants

In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.     

Alienation and drinking motivations among adolescent females.

188 female and 140 male 9th–12th graders in a small Wyoming community completed measures of alienation, drinking motivations, and positive social and personal psychological functions of drinking. Among females, problem-related drinking motivations correlated positively with alienation, whereas social-convivial motivations correlated negatively with this attribute. No comparable correlations were found among the males. It is concluded that important sex differences in the dynamics of problem drinking are worthy of additional study. (28 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A multi-institutional, multinational study of programming concepts using card sort data

Abstract: This paper presents a case study of the use of a repeated single‐criterion card sort with an unusually large, diverse participant group. The study, whose goal was to elicit novice programmers' knowledge of programming concepts, involved over 20 researchers from four continents and 276 participants drawn from 20 different institutions. In this paper we present the design of the study and the unexpected result that there were few discernible systematic differences in the population. The study was one of the activities of the National Science Foundation funded Bootstrapping Research in Computer Science Education project (2003).     

Characterizing vegetation indices derived from active and passive sensors

New ‘active’ sensors containing their own light source may provide consistent measures of plant and soil characteristics under varying illumination without calibration to reflectance. In 2006, an active sensor (Crop Circle) and various passive sensors were compared in a wheat (Triticum aestivum L., c.v. Chara) experiment in Horsham, VIC, Australia. The normalized difference vegetation index (NDVI) and soil-adjusted vegetation index (SAVI) were calculated from plot data with a range of canopy cover, leaf area and biomass. The active sensor NDVI and SAVI data were slightly less effective than corresponding passive sensor data at estimating green cover (r ²?=?0.80?0.90 vs. ～0.95). Passive sensor measurements showed strong non-linearity for estimating dry biomass and green leaf area index (GLAI), whereas SAVI calculated from the active sensor was linear (r ²?=?0.86 and 0.90). Scaling effects were not apparent when point, transect and plot areas were compared at the given level of spatial variation. Sensor height above the target confounded SAVI data probably due to differential irradiance from the light sources and the unbalanced effect of the ‘L’ factor within the algorithm. The active sensor was insensitive to light conditions (r ²?=?0.99 for cloudy vs. clear skies) and had no requirement for optical calibration.     

Redox control of exofacial protein thiols/disulfides by protein disulfide isomerase

Protein disulfide isomerase (PDI) facilitates proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum and is secreted by cells and associates with the cell surface. We examined the consequence of over- or underexpression of PDI in HT1080 fibrosarcoma cells for the redox state of cell-surface protein thiols/disulfides. Overexpression of PDI resulted in 3.6-4. 2-fold enhanced secretion of PDI and 1.5-1.7-fold increase in surface-bound PDI. Antisense-mediated underexpression of PDI caused 38-53% decreased secretion and 10-33% decrease in surface-bound PDI. Using 5,5'-dithio-bis(2-nitrobenzoic acid) to measure surface protein thiols, a 41-50% increase in surface thiols was observed in PDI-overexpressing cells, whereas a 29-33% decrease was observed in underexpressing cells. Surface thiol content was strongly correlated with cellular (r = 0.998) and secreted (r = 0.969) PDI levels. The pattern of exofacial protein thiols was examined by labeling with the membrane-impermeable thiol reactive compound, 3-(N-maleimidylpropionyl)biocytin. Fourteen identifiable proteins on HT1080 cells were labeled with 3-(N-maleimidylpropionyl)biocytin. The intensity of labeling of 11 proteins was increased with overexpression of PDI, whereas the intensity of labeling of 3 of the 11 proteins was clearly decreased with underexpression of PDI. These findings indicated that secreted PDI was controlling the redox state of existing exofacial protein thiols or reactive disulfide bonds.     

Identification and characterization of a lysis module present in a large proportion of bacteriophages infecting Streptococcus thermophilus

A lysis module encoded by the temperate bacteriophage phiO1205 was identified. This lysis module contains a lysin gene, designated lyt51, and two putative holin-encoding genes, designated lyt49 and lyt50. lyt51 encodes a lytic enzyme specifically directed against streptococcal cell walls. Similar to other phage-encoded lysins, Lyt51 appears to have a modular design in which the N-terminal portion corresponds to its enzymatic activity while the C-terminal region is responsible for its substrate binding specificity. The two putative holin-encoding genes, lyt49 and lyt50, located immediately upstream of lyt51, were identified on the basis of their homology to other identified holin-encoding genes. Expression of lyt49 or lyt50 in Escherichia coli was shown to cause cell death and leakage of the intracellular enzyme isocitrate dehydrogenase into the growth medium without apparent lysis of the cells. Southern blotting experiments demonstrated that at least one of the three components of the identified lysis module is present in all members of a large collection of bacteriophages, indicating that components of this lysis module are widespread among bacteriophages infecting Streptococcus thermophilus.