Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5-hen egg lysozyme complex and its individual components has been studied using continuum electrostatic calculations. Several residues of HyHEL-5 had pKa values shifted away from model values for isolated residues by more than three pH units. Shifts away from the model values were smaller for the residues of hen egg lysozyme. A moderate variation in the pKa values of the titratable groups was observed upon increase of the ionic strength from 0 to 100 mM, amounting to 1-2 pH units in most cases. Under physiological conditions, the net charge of HyHEL-5 was opposite that for hen egg lysozyme. Several residues, including those involved in the Arg-Glu salt bridges that have been proposed to be important in antibody-antigen binding, had pKa values that were changed significantly upon binding. The main titration event upon antibody-antigen binding appears to be loss of a proton from residue GluH50 of the Fv molecule. The limitations of our calculation methods and the role they might play in the design of antibodies for use in assays, sensors and separations are discussed.     

An outbreak of Shigella sonnei infection associated with consumption of iceberg lettuce

OBJECTIVES: This study compared the relative strength of the associations of a set of structural (social, economic, and political) variables and a set of health services variables with state-level infant, neonatal, and postneonatal mortality. It also examined whether health services mediate the relationships between structural variables and state-level infant, neonatal, and postneonatal mortality. METHODS: With the state as the unit of analysis, data for all 50 states were analyzed by means of multiple regression. RESULTS: Structural variables accounted for substantially more variance in infant, neonatal, and postneonatal mortality than health services variables, and health services variables were more strongly related to infant mortality than to neonatal or postneonatal mortality. When health services variables were controlled, the strengths of the associations between the structural variables and infant, neonatal, and postneonatal mortality were reduced but remained statistically significant. CONCLUSIONS: A substantial portion of the variance in state-level infant mortality is accounted for by states' structural characteristics, which are partially mediated by health services.     

Contractile responses and structure of small bronchi isolated from rats after 20 months' exposure to ozone

Short-term exposure to high concentrations of ozone has been shown to increase airway responsiveness in normal humans and in all laboratory animal species studied to date. While our knowledge concerning the pulmonary effects of single exposures to ozone has increased rapidly over recent years, the effects of repeated exposures are less understood. The goal of the present study was to determine whether airway responsiveness is increased after near-lifetime exposure to ozone. Airway segments representing approximately eighth generation airways were isolated from Fischer 344 rats of both genders that had been exposed for 6 hr per day, 5 days per week for 20 months to 0, 0.12, 0.5, or 1.0 parts per million (ppm) ozone. Circumferential tension development was measured in isolated airways in response to bethanechol, acetylcholine, and electrical field stimulation. Responsiveness of the airways to the contractile stimuli was described by the effective dose or frequency that elicited half-maximum contraction (ED50) and the maximum response. Since ozone exposure is associated with remodeling of peripheral airways, smooth muscle area was determined and tension responses were normalized to the area measurements. Before normalization of tension data to smooth muscle area, neither the ED50 nor maximum response of small bronchi to the contractile stimuli was altered after chronic ozone exposure. Smooth muscle area was greater in airways isolated from animals that had been exposed to 0.5 ppm ozone. After accounting for smooth muscle area, maximum responses of the small bronchi isolated from male rats were significantly reduced after 0.12 and 0.5 ppm ozone. Although not significant statistically, a similar trend was observed in airways isolated from female rats. These results suggest that the increase in airway responsiveness associated with acute ozone exposure does not persist during near-lifetime exposure. Although the mechanism responsible for the adaptation to the effects of O3 on airway responsiveness is unknown, the results indicate that smooth muscle cell function was compromised by the chronic exposure. The mechanism(s) responsible for mediating this effect and the relevance of these results to humans remains to be determined.     

Ornithine decarboxylase transformation of NIH/3T3 cells is mediated by altered epidermal growth factor receptor activity

Ornithine decarboxylase (ODC) has been shown to be oncogenic in transfected NIH/3T3 cells overexpressing the enzyme from a heterologous promoter. These cells, designated as NODC-2 cells, acquire proliferative properties associated with tumorigenic transformation such as loss of contact inhibition, decreased population doubling time, anchorage-independent growth, and tumor production in nude mice. At least one of these parameters, loss of contact inhibition, remains dependent on elevated ODC levels. We have used these cells to investigate the molecular mechanisms by which ODC overexpression drives cell transformation and to examine the involvement of other proto-oncogene products in this process. An interaction between ODC overexpression and the epidermal growth factor receptor (EGF-R) was suggested initially by the elevation of both basal (300%) and ligand-induced (457%) EGF-R tyrosine kinase activities in NODC-2 cells compared to similarly treated control NLK cells. Disruption of EGF-R mediated signal transduction in NODC-2 cells both by treatment with tyrphostin-25 or by transfection with a vector expressing a dominant negative EGF-R mutant resulted in reacquisition of contact-inhibited growth and suppression of anchorage-independent, clonogenic growth in soft agar. We conclude that ODC-induced transformation of NIH/3T3 cells is mediated, at least partly, by alterations in EGF-R signal transduction activity.     

The neuroprotective effect of the glycine site antagonist 3R-(+)-cis-4-methyl-HA966 (L-687,414) in a rat model of focal ischaemia

BACKGROUND: Cardiovascular conditioning reduces resting myocardial oxygen demand by lowering systolic blood pressure and heart rate. Lower myocardial oxygen demand at rest would be expected to be associated with a decrease in resting myocardial blood flow and, consequently, an increase in myocardial flow reserve as the ratio of hyperemic to resting blood flow. However, the effect of controlled exercise together with a low-lipid diet on myocardial blood flow and flow reserve has not been examined in humans. METHODS AND RESULTS: Myocardial blood flow at rest and after dipyridamole-induced hyperemia (0.56 mg/kg i.v.) was quantified with [13N]ammonia and positron emission tomography in 13 volunteers before and upon completion of a 6-week program of cardiovascular conditioning and a low-fat diet. Exercise capacity and serum lipid profiles were also assessed at the start and finish of the program. Eight normal volunteers of similar age not participating in the conditioning program served as a control group. Cardiovascular conditioning lowered the resting rate-pressure product (8859 +/- 2128 versus 7450 +/- 1496, P < .001), serum cholesterol (217 +/- 36 versus 181 +/- 26 mg/dL), LDL cholesterol (140 +/- 32 versus 114 +/- 24 mg/dL), and triglycerides (145 +/- 53 versus 116 +/- 33 mg/dL, all P < .05). Exercise tolerance (metabolic equivalent of the task, METs) improved significantly from 10.0 +/- 3.0 to 14.4 +/- 3.6 (P < .01). Resting blood flow decreased (0.78 +/- 0.18 versus 0.69 +/- 0.14 mL.g-1.min-1, P < .05), whereas hyperemic blood flow increased (2.06 +/- 0.35 versus 2.25 +/- 0.40 mL.g-1.min-1, P < .05), resulting in an improved myocardial flow reserve (2.82 +/- 1.07 versus 3.39 +/- 0.91, P < .05). Overall, the myocardial flow reserve was significantly related to exercise performance (METs). In the control group, no changes in resting rate-pressure product, serum cholesterol levels, exercise performance, resting or hyperemic myocardial blood flow, or flow reserve were observed. CONCLUSIONS: Short-term cardiovascular conditioning together with a low-fat diet results in an improved myocardial flow reserve by lowering resting blood flow and increasing coronary vasodilatory capacity. These changes are associated with an improved exercise capacity and may offer a protective effect in patients with coronary artery disease.     

Anti-hepatitis A virus antibody response elicited in mice by different forms of a synthetic VP1 peptide

To explore factors associated with longevity, we studied geographic distribution of centenarians in Japan, based on 1990 the population census. We calculated the proportion of centenarians from ratio of number of centenarian to that of population aged 65 years or older. Centenarians in Japan consisted of number 4,152 persons. By prefecture, Tokyo had the most centenarians (383), followed by Okinawa (193) and Fukuoka (151) prefectures. Fukui had the least (24), followed by Akita (26) and Ishikawa (29) prefectures. The proportion of centenarians in Japan was 21.6 (per 100,000 populations) in 1990. By prefecture, the highest proportion lived in Okinawa (133.8), whereas the fewest were found in Akita (8.9). The relationship between geographic distribution of centenarians and environmental factors and nutritional factors were analyzed. Correlation coefficients between proportion of centenarians and mean temperature, high quality of welfare work and of medical services, and having much leisure time were positively significant. As for nutritional factor, correlation coefficients between proportion of centenarians and protein (% of energy) was positively significant, while intake of total energy was negatively significant.     

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.