Quantitative immunoblotting assay of blood coagulation factor XII

The immunoblotting technique was applied to the study of Factor XII (F.XII) in plasma. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole plasma followed by electroblotting of the electropherograms to nitrocellulose (NC) membranes and immunologic detection by a double antibody technique was used. ¹²⁵-F.XII was transferred to the NC membrane in amounts proportional to the amount applied to the gel provided that a constant amount of carrier protein was present. Based on this, a quantitative assay was developed using either normal plasma or F.XII dilutions in F.XII-deficient plasma as standards. The measurement of F.XII antigen by immunoblotting was reproducible and gave values similar to those obtained by radial immunodiffusion. Two normal plasma pools contained 26 and 29 μ/ml of F.XII according to the immunoblotting assay. Compared to other immunoassays, immunoblotting has the advantage of directly estimating the apparent molecular weight (MW) of the protein of interest. Thus, we could confirm the normal apparent MW (80,000) of a F.XII-like molecule previously isolated from a cross reacting material (CRM)-positive F.XII-deficient plasma. None of eight CRM-negative F.XII-deficient plasmas showed an 80,000 MW immunoreactive molecule. However, five of these eight plasmas had a faint autoradiographic band at 115,000 MW that was similarly seen in only three out of 43 individual normal plasmas. The nature of this 115,000 MW band remains to be defined.     

Contribution of CNS neuropeptide (NPY, CRH, and beta-endorphin) alterations to psychophysiological abnormalities in anorexia nervosa

Patients with anorexia nervosa have neuroendocrine and behavioral alterations that starvation and weight loss are thought to cause, or contribute to, since they are reversed by weight restoration. We have found that anorexics have starvation-related disturbances of neuropeptide Y (NPY), corticotropin-releasing hormone (CRH), and beta-endorphin, as determined by their measurements in cerebrospinal fluid. The relationship between these neuropeptides and several symptoms in anorexia, together with findings in experimental animals, raise a possibility that changes in the activity of these neuropeptides contribute to neuroendocrine and behavioral alterations in anorexia. Specifically, a disturbance of central nervous system CRH activity is likely to be responsible for hypercortisolemia, while a disturbance of central nervous system NPY may contribute to amenorrhea. In addition, disturbances of these neuropeptides could contribute to other symptoms such as increased physical activity, hypotension, reduced sexual interest, depression, and pathological feeding behavior.     

Diplotypes of the human serotonin 1B receptor promoter predict growth hormone responses to sumatriptan in abstinent alcohol-dependent men.

BACKGROUND: Some studies have associated alcohol dependence (AD) with the human serotonin (5-HT)(1B) receptor (HTR1B). This investigation explored the functional responsivity of HTR1B in abstinent AD men using a sumatriptan challenge, while measuring genetic heterogeneity in the HTR1B promoter. METHODS: Abstinent AD men (n = 27) and abstinent men without any alcohol use disorder (n = 19) were administered 6 mg of sumatriptan succinate, subcutaneously. Plasma samples collected over the following 2 hours were assayed for growth hormone (GH) concentrations. His DNA was genotyped for the A-161T and T-261G polymorphisms of the HTR1B promoter and diplotypes determined. RESULTS: Integrated GH responses were predicted by interactions of AD and promoter diplotypes, as well as subject ethnicity. The final model accounted for nearly 35% of the variance in GH responses. Post hoc evaluation revealed that AD was associated with a blunting of GH secretion only among individuals with the most common HTR1B diplotype (TT/TT). CONCLUSIONS: A blunting of GH responses in abstinent AD men was observed only among those with the most common HTR1B promoter diplotype. Less common promoter diplotypes appeared protective. Controlling for genetic background is a useful augmentation of case-control pharmacological challenge strategies designed to elucidate the psychobiology of AD and other complex disorders.     

Vasoactive intestinal peptide and bipolar affective illness. Evidence for an effect of lithium

In an attempt to evaluate the role of VIP in affective disorder, measurements of lymphocyte VIP receptors, and plasma and CSF VIP levels were made in unmedicated and lithium-treated euthymic bipolars and controls. Lithium decreased plasma (P = 0.01) and CSF (P = 0.05) VIP levels and increased the affinity (decreased the KD) of the VIP lymphocyte receptor (P less than 0.01). This effect may be relevant to the psychotropic action of lithium in manic-depressive illness.     

Linkage analysis of anorexia and bulimia nervosa cohorts using selected behavioral phenotypes as quantitative traits or covariates.

To increase the likelihood of finding genetic variation conferring liability to eating disorders, we measured over 100 attributes thought to be related to liability to eating disorders on affected individuals from multiplex families and two cohorts: one recruited through a proband with anorexia nervosa (AN; AN cohort); the other recruited through a proband with bulimia nervosa (BN; BN cohort). By a multilayer decision process based on expert evaluation and statistical analysis, six traits were selected for linkage analysis (1): obsessionality (OBS), age at menarche (MENAR), and anxiety (ANX) for quantitative trait locus (QTL) linkage analysis; and lifetime minimum body mass index (BMI), concern over mistakes (CM), and food-related obsessions (OBF) for covariate-based linkage analysis. The BN cohort produced the largest linkage signals: for QTL linkage analysis, four suggestive signals: (for MENAR, at 10p13; for ANX, at 1q31.1, 4q35.2, and 8q13.1); for covariate-based linkage analyses, both significant and suggestive linkages (for BMI, one significant [4q21.1] and three suggestive [3p23, 10p13, 5p15.3]; for CM, two significant [16p13.3, 14q21.1] and three suggestive [4p15.33, 8q11.23, 10p11.21]; and for OBF, one significant [14q21.1] and five suggestive [4p16.1, 10p13.1, 8q11.23, 16p13.3, 18p11.31]). Results from the AN cohort were far less compelling: for QTL linkage analysis, two suggestive signals (for OBS at 6q21 and for ANX at 9p21.3); for covariate-based linkage analysis, five suggestive signals (for BMI at 4q13.1, for CM at 11p11.2 and 17q25.1, and for OBF at 17q25.1 and 15q26.2). Overlap between the two cohorts was minimal for substantial linkage signals.     

Platelet activation in psoriasis

Recent epidemiological studies have suggested that psoriasis represents a risk factor for thrombotic vascular diseases. In order to evaluate the possible role of hemostatic changes in the development of thrombotic episodes in psoriasis, some parameters of the hemostatic "balance" were investigated in 22 male psoriatic patients and compared to those of 22 male control subjects. Incidence of known risk factors for vascular diseases (diabetes, hypertension, smoking, dyslipidemia) was comparable in the two study groups. There were no statistically significant differences in platelet count, circulating platelet aggregates, platelet production of malondialdehyde (MDA), total plasma antithrombin and fibrinolytic activities. In patients with psoriasis the incidence of spontaneous platelet hyperaggregability and plasma levels of beta-thromboglobulin were significantly higher than in control subjects. Platelet regeneration time, measured as MDA recovery after aspirin ingestion, was significantly shorter in psoriatic patients. These data suggest that an in vivo platelet activation occurs in patients with psoriasis and could contribute to the development of thrombotic complications. The release of mitogenic and inflammatory substances by activated platelets may play a role in the histogenesis of psoriatic lesions.     

“In vitro” and “ex vivo” effects of picotamide,a combined thromboxane A2-synthase inhibitor and -receptor antagonist,on human platelets

Summary Picotamide (G 137), a new non prostanoid inhibitor of in vitro arachidonic acid induced platelet aggregation, has been further characterized in in vitro and ex vivo studies.When whole blood was activated with collagen in the presence of picotamide 5×10^–4 M, thromboxane B₂ production was decreased, and 6-keto-PGF₁ generation was significantly increased, suggesting a reorientation of platelet endoperoxide metabolism following blockade of thromboxane synthetase. Picotamide also inhibited platelet aggregation and clot retraction induced by the endoperoxide analogue U46619 in human platelets, indicating thromboxane A₂-receptor antagonism, possibly of competitive nature.A single oral dose of picotamide 1 g in 24 healthy volunteers produced a significant inhibition of collagen, arachidonic acid and U46619-induced platelet aggregation. Serum levels of thromboxane B₂ were also reduced.Chronic administration of picotamide 1.2 g/d to patients with vascular disease resulted in a prompt and persistent fall in their increased plasma levels of -thromboglobulin.The results indicate that picotamide is a combined thromboxane B₂-synthetase inhibitor and thromboxane A₂-receptor antagonist in human platelets, and that it may prove useful as an antithrombotic agent.     

High-resolution magnetic resonance and volume rendering of the labyrinth

Our aim was to verify the feasibility of volume rendering (VR) of high-resolution magnetic resonance (HR-MR) data sets of the labyrinth. We retrospectively reviewed the HR-MR data sets of 16 consecutive patients with no MR evidence of labyrinthine pathology. High-resolution MR data sets were obtained by means of a 3D T2-weighted FSE sequence with the use of a 3-in. circular surface coil for signal reception, and processed with a high-end workstation. Two reviewers performed separately VR of the labyrinth by selecting the signal intensity interval for attribution of opacity and transparency. Concerning the time taken for definition of the volume of interest, the two observers needed, respectively, 28.9 and 33.1 min (SD ± 8.7–9.5 min), whereas the time taken for VR was respectively, 26 and 33.2 min (SD ± 8.8–8.9 min). Concerning the selection of the signal intensity interval, the two observers had, respectively, 86.4 and 88.7 mean lower threshold (SD ± 34.5–33.5), 488.9 and 495.4 mean upper threshold (SD ± 56.3–53.8). In our experience, we have found VR of HR-MR to offer a reliable and reproducible technique for producing 3D representations of the labyrinth. The VR algorithms use all data within the imaging volume and optimize the dynamic range ascribed to the object being visualized. Received: 9 October 1998; Revised: 14 January 1999; Accepted: 30 June 1999     

Confirmation of a major QTL influencing oral morphine intake in C57 and DBA mice using reciprocal congenic strains.

C57BL/6 (B6) and DBA/2 (D2) mice exhibit disparate behavior when tested for voluntary morphine intake in a two-bottle choice drinking paradigm with B6 mice consuming 10 times more drug than D2 mice. Previous genetic mapping studies identified a locus, Mop2, on the proximal part of chromosome 10 that explained over half of the genetic variance in this mouse model of opioid self-administration. We constructed a set of reciprocal congenic strains between B6 and D2 mice in which the proximal portion of chromosome 10 has been introgressed from one strain onto the background of the other. We tested mice from this pair of reciprocal strains together with progenitor B6 and D2 mice in a two-bottle choice drinking paradigm with morphine and quinine. The results showed that introgression of chromosome 10 alleles from the B6 strain onto a D2 genetic background increased voluntary morphine intake four-fold compared to progenitor D2 mice. Preference for morphine was also increased significantly in D2.B6-Mop2 mice compared to progenitor D2 mice. Conversely, introgression of chromosome 10 alleles from the D2 strain onto a B6 genetic background decreased morphine intake by half compared to progenitor B6 mice in B6.D2 -Mop2 mice; however, high morphine preference was maintained in this congenic strain most likely due to strong quinine aversion. When quinine was eliminated from the control bottle, morphine preference in B6.D2-Mop2 mice was decreased significantly relative to B6 and D2.B6-Mop2 mice. Overall, these data confirm the existence of a gene(s) on chromosome 10 proximal to D10Mit124 that has a strong influence on the difference in morphine drinking behavior between B6 and D2 mice.