Cross-linking of atrial natriuretic peptide to binding sites in rat olfactory bulb membranes

Binding sites for 125I-atrial natriuretic peptide (ANP)2 in rat olfactory bulb membranes have been studied using pharmacological and biochemical methods. Various unlabeled ANP-related peptides were tested for the ability to inhibit the binding of the radioligand in membrane binding assays. ANP(92-126) and ANP(99-126) were the most potent inhibitors tested, both exhibiting an IC50 value of 0.40 nM. ANP(103-126) and ANP(103-123) were 3 and 70 times less potent, respectively. ANP(111-126) was unable to inhibit the binding of the radioligand at a concentration of 1 microM. Several peptides unrelated to ANP were unable to inhibit the binding of the radioligand to rat olfactory bulb membranes. Membranes labeled with 125I-ANP were incubated with cross-linking agents and subjected to SDS-PAGE followed by autoradiography. A band possessing an apparent molecular mass of 116 kDa was identified. The labeling of this band was progressively decreased by increasing concentrations of unlabeled ANP(99-126) (IC50 = 0.6 nM) and by several other ANP-related peptides at nanomolar concentrations. For comparison purposes, ANP binding sites in rat aorta membranes were labeled with 125I-ANP and cross-linked using identical techniques. Three bands possessing molecular masses of 120, 72, and 62 kDa were identified. These results indicate that the ANP binding site in rat olfactory bulb membranes displays pharmacological and biochemical properties similar to peripheral ANP receptors.     

The influence of alcohol on the outcome of automated static perimetry

It is well known that perimetric findings fluctuate within a single examination. There is additional fluctuation between perimetric examinations. The cause of this fluctuation is not yet fully understood, but such things as changes in attention, patient cooperation, or drugs have been discussed. To study such possible factors, we carried out perimetry on subjects who had consumed alcohol and who had not. The results indicate that alcohol, at a blood concentration of approximately 0.08%, barely influences the results of static automated perimetry. Differential light sensitivity remained unchanged by alcohol at all eccentricities tested. A decrease in the ability to cooperate was manifested by a significant higher score of false-positives in catch trials. There was also a tendency toward an increase in false-negative responses in catch trials, an increase in the number of stimuli presentations required, and higher short-term fluctuation. Lack of the influence of alcohol on the differential light threshold does not necessarily mean that alcohol has no influence on visual function. It indicates, however, that differential light sensitivity, as measured with the automated perimeter Octopus, is not influenced by moderate alcohol ingestion.     

Experimental demonstration of the immunogenicity of silicone-protein complexes

Although silicones, as a class, are nontoxic in animal and tissue studies, implanted silicone prostheses and medical devices are associated with various local and systemic host inflammatory reactions. They also have been associated with a form of autoimmune disease. To test the hypothesis that silicones may evoke an immunologically mediated inflammatory reaction, 10 guinea pigs were stimulated for 1 month with intraperitoneal injections of sterile medical-grade silicone oil admixed with homologous serum and complete Freund's adjuvant. Ten controls were stimulated with saline. Four additional animals were passively sensitized with splenic homogenates from four sensitized animals. Intradermal antigenic challenges consisted of silicone-homologous serum, pure silicone, saline-homologous serum, and purified protein derivative. Cutaneous reaction patterns were graded grossly and microscopically. Silicone-serum and purified protein derivative antigens evoked three to four times greater palpable lesions in all 10 actively and all 4 passively sensitized animals at approximately 24 h compared to controls. Biopsies showed a moderate to marked lymphocytic infiltrate. Control sites and naive animals showed only edema at the challenge sites. The data suggest that silicone-protein complexes are potentially immunogenic.     

Effect of goldenseal (Hydrastis canadensis) and kava kava (Piper methysticum) supplementation on digoxin pharmacokinetics in humans.

Phytochemical-mediated modulation of P-glycoprotein (P-gp) and other drug transporters may give rise to many herb-drug interactions. Serial plasma concentration-time profiles of the P-gp substrate, digoxin, were used to determine whether supplementation with goldenseal or kava kava modified P-gp activity in vivo. Twenty healthy volunteers were randomly assigned to receive a standardized goldenseal (3210 mg daily) or kava kava (1227 mg daily) supplement for 14 days, followed by a 30-day washout period. Subjects were also randomized to receive rifampin (600 mg daily, 7 days) and clarithromycin (1000 mg daily, 7 days) as positive controls for P-gp induction and inhibition, respectively. Digoxin (Lanoxin, 0.5 mg) was administered p.o. before and at the end of each supplementation and control period. Serial digoxin plasma concentrations were obtained over 24 h and analyzed by chemiluminescent immunoassay. Comparisons of area under the curve (AUC)((0-3)), AUC((0-24)), C(max,) CL/F, and elimination half-life were used to assess the effects of goldenseal, kava kava, rifampin, and clarithromycin on digoxin pharmacokinetics. Rifampin produced significant reductions (p < 0.01) in AUC((0-3)), AUC((0-24)), CL/F, t(1/2), and C(max), whereas clarithromycin increased these parameters significantly (p < 0.01). With the exception of goldenseal's effect on C(max) (14% increase), no statistically significant effects on digoxin pharmacokinetics were observed following supplementation with either goldenseal or kava kava. When compared with rifampin and clarithromycin, supplementation with these specific formulations of goldenseal or kava kava did not appear to affect digoxin pharmacokinetics, suggesting that these supplements are not potent modulators of P-gp in vivo.     

Probing insulin's secondary structure after entrapment into alginate/chitosan nanoparticles.

The aim of the present study was to probe the structural integrity of insulin after being entrapped into chitosan/alginate nanoparticles produced by ionotropic polyelectrolyte pre-gelation. By manipulating the alginate:chitosan mass ratio and the pH during nanoparticle production, desired nanoparticles with a mean size of 850 (+/-88)nm and insulin association efficiency of 81 (+/-2)% were obtained. Insulin secondary structure was assessed by Fourier transform infrared (FTIR) and circular dichroism (CD) after entrapment into nanoparticles and after release from the particles under gastrointestinal simulated conditions. FTIR second-derivative spectra and area-overlap compared to an insulin standard confirmed that no significant conformational changes of insulin occurred in terms of alpha-helix and beta-sheet content. Far-UV-CD spectra corroborated the preservation of insulin structure during the nanoparticle production procedure. The presented nanoparticulate system is a promising carrier for insulin oral delivery since it preserves insulin structure and therefore also, potentially, its bioactivity.