Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor

The interactions between ligands containing the recognition sequence arginine-glycine-aspartic acid (RGD) and integrin receptors are important in many cell-cell and cell-protein interactions. The platelet contains five integrin receptors and they contribute significantly to platelet adhesion and aggregation. To investigate the RGD binding domains on platelet integrins, we immobilized a series of RGD peptides containing variable numbers of glycine residues [(G)n-RGDF] on polyacrylonitrile beads and evaluated the ability of the beads to interact with platelets. With native platelets, virtually no interaction occurred with G1-RGDF beads, but the interactions increased as the number of glycine residues increased, plateauing with the G9- RGDF and G11-RGDF beads. ADP pretreatment enhanced the interactions with all of the beads, whereas prostaglandin E1 pretreatment eliminated the interactions with the shortest peptide beads, but only partially inhibited interactions with the longer peptide beads. Monoclonal antibodies to glycoprotein (GP) IIb/IIIa were most effective in inhibiting the interactions, but antibodies to GPIIb/IIIa with similar inhibitory effects on fibrinogen binding varied dramatically in their ability to inhibit the interaction between platelets and immobilized RGD peptides. Our data indicate that the majority of RGD binding sites on GPIIb/IIIa can be reached by peptides that extend out approximately 11 to 32 A from the surface of the bead, and these results are in accord with the dimensions of integrin receptors deduced from electron microscopy. Activation of GPIIb/IIIa facilitates the interactions, but platelet inhibition fails to eliminate the interactions with the longer peptide beads, suggesting that access to the RGD binding site on at least a fraction of the GPIIb/IIIa receptors is always possible for preferred ligands. Finally, we found that the G3-RGDF peptide beads were uniquely sensitive to the activation state of the GPIIb/IIIa receptor.     

Localization and visualization of pulmonary emboli with radiolabeled fibrin-specific monoclonal antibody

Indium-111-labeled monoclonal antibody 64C5 specific for the beta-chain of fibrin monomer was used to image canine (n = 6) experimental pulmonary emboli (at least one barium-thrombin and one copper-coil induced clot per dog). Uptake of 111In-64C5 and 125I-control-DIG26-11 were compared in 10 clots (7 barium-thrombin and 3 copper-coil) identified in the lungs. There was no difference in the blood clearance of 111In-64C5 and 125I-DIG26-11. Uptake of 111In-64C5 (0.183 +/- 0.105, mean %ID/g) was greater than 125I-DIG26-11 (0.024 +/- 0.025) in pulmonary clots (p less than 0.001). Mean thrombus to blood ratios at 24 hr were 6.78:1 for 64C5 and 0.57:1 for DIG26-11. The clots visualized in vivo were larger (0.315 +/- 0.381 g) than clots not visualized (0.089 +/- 0.098). Negative images were recorded in three dogs with pulmonary emboli, injected with 111In-labeled control monoclonal antibody 3H3. These data suggest that 111In-labeled antifibrin can detect large pulmonary emboli in vivo.     

The effect of phenothiazines upon maintenance of membrane integrity in the cultured myocardial cell

The cultured myocardial cell provides a defined model for examining factors which are responsible for maintaining cellular viability and sarcolemmal integrity. Our data indicates that the spontaneous loss of myocyte membrane integrity is a calcium-dependent process and thus provides a method for examining the mechanism through which calcium exerts this effect. Antimyosin antibody staining and propidium iodide uptake were used to quantitate membrane integrity. The integrity of the cell membrane was inversely related to the calcium concentration in the culture medium. This loss of membrane integrity was calmodulin-dependent as demonstrated by the following: phenothiazines (trifluoperazine greater than chlorpromazine greater than promethazine) and structurally dissimilar calmodulin-inhibitors prevented the formation of sarcolemmal defects at concentrations similar to those known to inhibit calmodulin; phenothiazines and calcium demonstrated a competitive interaction with respect to this effect on membrane integrity. Electron microscopy confirmed the integrity of the sarcolemma of the cells exposed to high phenothiazine concentrations although metabolic alterations occurred in these cells as evidenced by an increased membrane permeability to the low molecular weight probe propidium iodide, degenerative changes in the fine structure of the mitochondria, the accumulation of autophagic vacuoles in the cytoplasm and the loss of contractile ability. These findings indicate that calmodulin inhibitory compounds are capable of preserving the membrane integrity of cardiac myocytes, interfering with a calcium-dependent process that is associated with the spontaneous attrition of these cells in culture. Significant intracellular alterations appear at high doses of these agents even while the sarcolemma is free of gross defects.     

Serum C-reactive protein is a useful marker for congenital infection

Serial quantitative measurements of C-reactive protein (CRP) were performed by reaction rate nephelometry on cord blood and scrum of 70 babies (60 preterm and 10 full term). There were 41 babies born to mothers with no risk factors for bacterial infection (Group 1) and 29 babies born to mothers with risk factors (Group 2), Maternal risk factors for congenital infection were: a history of prolonged rupture of membranes, maternal treatment with antibiotics, chorioamnionitis or positive cultures from high vaginal swabs. Twelve babies had raised CRP levels (>10 mg/1), 2 from Group 1 and 10 from Group 2, 9 of whom were subsequently found to have bacterial infections. One of the 2 babies without suspected infection had severe birth asphyxia. Specificity, sensitivity, positive and negative predictive values were calculated for CRP levels and total white cell counts (TWCC). Within Group 2, the specificity of raised CRP for diagnosis of congenital bacterial infection was 95% and the sensitivity was 100%, compared to the specificity of TWCC 95% and sensitivity 55%. CRP can be used as a marker for congenital bacterial infection and appears to be more sensitive than TWCC.     

Sleep EEG evidence of sex differences in adolescent brain maturation

STUDY OBJECTIVES: The steep decline in slow-wave (delta) electroencephalogram (EEG) intensity across adolescence is a prominent feature of late brain maturation. As a first step in determining whether the adolescent delta decline is similar in both sexes, we compared cross-sectional sleep EEG data from 9- and 12-year-old boys and girls. DESIGN: All-night EEG recordings, 6 months apart, were conducted on each subject. SETTING: EEG was recorded in the subjects' homes. PARTICIPANTS: Thirty-two 9-year-olds and 38 12-year-olds are enrolled in a 4-year longitudinal study of adolescent sleep. There are equal numbers of each sex in both age cohorts. INTERVENTIONS: N/A. MEASUREMENTS: Using ambulatory recorders, EEG was recorded in the subjects' homes on their normal sleep schedule. For each of the 2 semi-annual recording periods, data from the 10 subjects from each age-sex group with the cleanest (fewest artifacts) signals were selected for crosssectional comparisons of visual scoring and EEG variables. All artifact-free 20-second non-rapid eye movement epochs were analyzed with power spectral and period-amplitude analysis. RESULTS: In the 12-year-old cohort, delta power per minute was 37% higher in boys than girls. The 9-year-old cohort showed no sex difference. A second recording 6 months later produced similar results. CONCLUSION: These cross-sectional data indicate that girls begin the steep adolescent decline in slow-wave EEG earlier than boys. We hypothesize that this reflects an earlier onset of adolescent synaptic pruning in females.     

Relationship of alcohol intake and sex steroid concentrations in blood in pre- and post-menopausal women: the European Prospective Investigation into Cancer and Nutrition

Objective Women with a moderate intake of alcohol have higher concentrations of sex steroids in serum, and higher risk of developing breast cancer, compared to non-drinkers. In the present study, we investigate the relationships between alcohol consumption and serum levels of sex steroids and sex-hormone binding globulin (SHBG) in 790 pre- and 1,291 post-menopausal women, who were part of the European Prospective Investigation into Cancer and Nutrition (EPIC). Methods Serum levels of testosterone (T), androstenedione (Δ₄), dehydroepiandrosterone sulphate (DHEAS), estrone (E₁), estradiol (E₂) and SHBG were measured by direct immunoassays. Free T (fT) and free E₂ (fE₂) were calculated according to mass action laws. Current alcohol intake exposure to alcohol was assessed from dietary questionnaires. Results Pre-menopausal women who consumed more than 25 g/day of alcohol had about 30% higher DHEAS, T and fT, 20% higher Δ₄ and about 40% higher E₁, concentrations compared to women who were non-consumers. E₂, fE₂ and SHBG concentrations showed no association with current alcohol intake. In post-menopausal women, DHEAS, fT, T, Δ₄, and E₁ concentrations were between 10% and 20% higher in women who consumed more than 25 g/day of alcohol compared to non-consumers. E₂ or fE₂ were not associated with alcohol intake at all. SHBG levels were about 15% lower in alcohol consumers compared to non-consumers. Conclusion This study supports the hypothesis of an influence of alcohol intake on sex hormone concentrations in blood. The EPIC study was funded by ‘‘Europe Against Cancer’’ Programme of the European Commission (SANCO); Ligue contre le Cancer (France); Société 3M (France); Mutuelle Générale de I’éducation Nationale; Institut National de la Santé et de la Recherche Médicale (INSERM); German Cancer Aid; German Cancer Research Center; German Federal Ministry of Education and Research; Danish Cancer Society; Health Research Fund (FIS) of the Spanish Ministry of Health (RCESP-C03/09); the participating regional governments and institutions of Spain; Cancer Research UK; Medical Research Council, UK; the Stroke Association, UK; British Heart Foundation; Department of Health, UK; Food Standards Agency, UK; the Wellcome Trust, UK; Greek Ministry of Health; Greek Ministry of Education; Italian Association for Research on Cancer (AIRC); Dutch Ministry of Public Health, Welfare and Sports; Dutch Ministry of Health; Dutch Prevention Funds; LK Research Funds; Dutch ZON (Zorg Onderzoek Nederland); World Cancer Research Fund (WCRF); Swedish Cancer Society; Swedish Scientific Council; Regional Government of Skane, Sweden; Norwegian Cancer Society; Specific study results of this case-control study nested within EPIC, presented in this paper, were obtained with financial support from the National Cancer Institute (USA) [grant nr1U01CA98216-01].