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The conformation and orientation of proteins immobilised on synthetic materials determine their ability to bind their antigens and thereby the sensitivity of the microarrays and biosensors employing them. Plasma immersion ion implantation (PIII) of polymers significantly increases both their wettability and protein binding capacity. This paper addresses the hypothesis that a PIII treated polymer surface modifies the native protein conformation less significantly than a more hydrophobic untreated surface and that the differences in surface properties also affect the protein orientation. To prove this, the orientation and conformation of rat anti-mouse CD34 antibody immobilized on untreated and PIII treated polycarbonate (PC) were investigated using ToF-SIMS and FTIR-ATR spectroscopy. Analysis of the primary structure of anti-CD34 antibody and principal component analysis of ToF-SIMS data were applied to detect the difference in the orientation of the antibody attached to untreated and PIII treated PC. The difference in the antibody conformation was analysed using deconvolution of the Amide I peak (in FTIR-ATR spectra) and curve-fitting. It was found that compared to the PIII treated sample, the antibody immobilized on the untreated PC sample has a secondary structure with a lower fraction of β-sheets and a higher fraction of α-helices and disordered fragments. Also, it was found that anti-CD34 antibody has a higher tendency to occur in the inactive 'tail-up' orientation when immobilized on an untreated PC surface than on a PIII treated surface. These findings confirm the above hypothesis., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Buprenorphine and naloxone sublingual (s.l.) dose formulations may decrease parenteral buprenorphine abuse. We evaluated pharmacologic interactions between 8 mg s.l. buprenorphine combined with 0, 4, or 8 mg of naloxone in nine opiate-dependent volunteers stabilized on 8 mg s.l. buprenorphine for 7 days. Combined naloxone and buprenorphine did not diminish buprenorphine's effects on opiate withdrawal nor alter buprenorphine bioavailability. Opiate addicts stabilized on buprenorphine showed no evidence of precipitated opiate withdrawal after s.l. buprenorphine-naloxone combinations. Buprenorphine and naloxone bioavailability was approximately 40 and 10%, respectively. Intravenous buprenorphine and naloxone produced subjective effects similar to those of s.l. buprenorphine and did not precipitate opiate withdrawal.

A gas chromatographic method for the determination of levorphanol in human plasma is described. The method utilizes extractive alkylation with tetrabutylammonium cation as the phase-transfer catalyst and pentafluorobenzyl bromide as the alkylating agent, and employs a structural analog, d-3-hydroxy-N-ethylmorphinan, as the internal standard. The pentafluorobenzyl ethers formed are separated by capillary gas chromatography and detected by electron capture. The method has good precision and accuracy for concentrations ranging from 0.25 ng/ml to 100 ng/ml and has been used to measure plasma concentrations as part of a study to evaluate the management of chronic neuropathic pain with levorphanol.

Although allergic persons can react to foods containing trace quantities of unlabeled or unintended food allergens, there are few data available on the quantities of these allergens required to evoke allergic symptoms. We report a milk-allergic 3-year-old boy who experienced throat itching, facial angioedema, and vomiting within 20 min of ingesting 4 to 6 oz (ca. 113.4 to 170.1 g) of lemon sorbet. Subsequent analysis of two sorbet samples provided by the parents and a third sample purchased locally by the investigators revealed trace quantities of milk allergens, whey protein (8.8 micrograms/ml), or lactose (200 ppm). The quantity of whey protein ingested was estimated to be 120 to 180 micrograms (equivalent to 23 to 24 microliters of milk). All three sorbet samples had been manufactured in the same plant within a 4-month period; the equipment used to produce and package the sorbet was also used to produce and package ice cream. No milk allergen or whey protein was detected in 38 other marketplace sorbet samples submitted by the manufacturer for testing. We concluded that trace quantities of whey proteins (< 200 micrograms) can elicit systemic reactions in exquisitely milk-allergic individuals. Such individuals should avoid eating frozen desserts prepared using equipment also used for producing or packaging ice cream, unless manufacturers can demonstrate unequivocally that their cleaning practices are sufficient to prevent milk contamination. Adequate tests are not currently available to food manufacturers but are under development.

Buprenorphine is undergoing clinical trials for the treatment of opiate addiction. Although the abuse liability of sublingual buprenorphine is low, reports of intravenous abuse have appeared. This study describes the physiologic and subjective effects of intravenously administered buprenorphine and naloxone given alone and in combination to methadone-maintained patients (40-60 mg/day). On four separate occasions at least 1 day apart, 6 subjects were administered either 0.2 mg buprenorphine, 0.1 mg naloxone, 0.2 mg buprenorphine and 0.1 mg naloxone in combination, or placebo. One male subject quit the experiment after three sessions because of excessive opiate withdrawal. Buprenorphine produced no significant physiologic or subjective effects. Naloxone produced marked opiate withdrawal symptoms. Buprenorphine in combination with naloxone produced characteristic physiologic and subjective opiate antagonist-like symptoms and signs. The parenteral abuse potential of the buprenorphine and naloxone combination is discussed.

Unlabelled: STUDY OBJECTIVES AND DESIGN: Arterial vasoconstriction is thought to play a role in the etiology of cocaine-induced cardiovascular complications, but little is known about the immediate effects of cocaine on the thoracic aorta and coronary arteries. To examine these effects, we used transesophageal echocardiography to examine the thoracic aorta and coronary arteries before and immediately after intravenous (i.v.) cocaine (1.2 mg/kg) in 15 subjects., Measurements and Results: Immediately after cocaine infusion, average heart rate, systolic BP, and double product were increased compared with baseline (22%, 15%, 35%, respectively). There was no significant change in the diameters of the ascending aorta (27.5 vs 27.1 mm; p = 0.85), the descending aorta (19.8 vs 20.4 mm; p = 0.62), or the left main coronary artery (4.3 vs 4.7 mm; p = 0.15). However, there was a trend for an increase in coronary blood flow immediately after cocaine (226 vs 309 mL/min; p = 0.10)., Conclusions: We conclude that in the 15 subjects studied, there was no evidence of thoracic aorta of coronary artery vasoconstriction immediately after i.v. cocaine. Instead, we found that the diameters of the thoracic aorta and the left main coronary artery were unchanged, and that there was a trend for augmentation of coronary artery blood flow.

This controlled study tested the efficacy of the selective serotonergic reuptake inhibitor fluoxetine in the out-patient treatment of primary crack cocaine dependence. Thirty-two subjects were randomly assigned, 16 in each group, to placebo or fluoxetine, 40 mg/day, in a double-blind controlled trial over a 12-week period. Outcome measures included quantitative urine benzoylecgonine concentration, self-reports of cocaine use and craving, and treatment retention. Subjects assigned to fluoxetine were retained in treatment significantly longer than those on placebo: a median of 11 weeks compared to 3 weeks (logrank test, P < 0.001). Because of the poor retention in the placebo group, between-groups comparisons of outcome were limited to the first 6 weeks of treatment. No differences in cocaine use or craving were found between the two groups over weeks 1 to 6. The significant improvement in retention associated with fluoxetine may support further study of this medication in the treatment of cocaine dependence.

A gas chromatographic method for the simultaneous determination of methamphetamine and its metabolite amphetamine in human plasma and urine is described. The method utilizes reductive alkylation with propionaldehyde and sodium borohydride to produce N-propyl derivatives, which have excellent chromatographic properties. Structural analogs of the analytes, p-methylmethamphetamine and p-methylamphetamine, are used as internal standards. The method has good precision and accuracy for concentrations ranging from less than 10 ng/ml to 5000 ng/ml and has been used to measure plasma concentrations as part of a pharmacokinetic/pharmacodynamic study of methamphetamine in humans.

Protein degradation rates are decreased in some transformed cells of mesenchymal origin. We have tested the generality of this phenomenon and evaluated the role of the lysosomes in this down-regulation. To this end we have compared the induction of lysosomal protein degradation among normal, transformed (BEAS-2B), and transformed tumorigenic (BZR, Calu-1) human bronchial epithelial cells in culture. Serum and/or nutrient deprivation, cell confluency, and Ca2+ were used to modulate lysosomal protein degradation. Protein degradation and synthesis were determined by the release or incorporation of [14C]valine in the cells. Autophagic degradation of cytoplasm by lysosomes was evaluated by ultrastructural morphometry. Basal protein degradation was lower (27%) in two of the transformed cell lines (BEAS-2B and BZR). Incorporation of [14C]valine label was raised approximately 4-fold in the transformed cells. Nutrient deprivation stimulated protein degradation equally (2-fold) in transformed and normal cells. Postconfluency increased (1.5-fold) basal protein degradation in Calu-1 cells and a marked enhancement (4-fold) of degradation occurred during nutrient deprivation. Culture of normal human bronchial epithelial cells in high Ca2+ caused phenotypic changes and increased (30%) the degradation of protein induced by nutrient deprivation. In Calu-1, high Ca2+ caused only phenotypic changes. The volume density (Vd) of autophagic vacuoles and dense bodies in the transformed cells was lower under basal conditions but increased markedly during nutrient deprivation. A marked accumulation of lysosomes also occurred in transformed cells during postconfluency. We conclude that cell transformation lowers basal protein degradation in some human epithelial cells. Lysosomal proteolysis of transformed cells is not down-regulated and can be markedly enhanced during nutritional deprivation by the autophagic degradation pathway.

A rapid and sensitive gas chromatographic method is described for the determination of cocaine in plasma. A close structural analogue of cocaine, m-toluylecgonine methyl ester, is used as an internal standard. The simplicity of the extraction scheme and the use of an automatic sampler makes the method convenient for large numbers of samples generated in pharmacokinetic studies.

The zeta (zeta) and various gamma (gamma) chains of Hb Portland I have been separated and isolated from blood samples obtained from neonates with hydrops fetalis due to homozygous alpha-thalassemia. By using developers containing acetonitrile, methanol, and potassium phosphate and either an analytical (3.9 mm x 30 cm) or a preparative (7.8 mm x 30 cm) muBondapak C-18 column (Waters), globin chains from 200 micrograms to 5.0 mg have been isolated in pure forms. Analytical and preparative procedures using short (50-min duration) and extended (186-min duration) gradient programs have been developed. In addition to the type of column and developer conditions, the following factors are found to be important: (a) preparation of sample, (b) sample loading, and (c) cleaning of the column. Preliminary studies indicate that the yield ranges from 40 to 60% depending on the type of globin sample and the age of the column. This procedure also permits the separation of alpha, beta, and various gamma globin chains from fetal and adult samples.

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated alpha-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity (P 1/2 = 0.60 and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect (-delta log P 1/2/pH (7 to 8) = 0.34 and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l' = 5.6 X 10(6) M-1 S-1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l' = 2.6 X 10(6) M-1 S-1), whereas Hb Wayne Asp showed heterogeneous binding (l' = 6.1 X 10(6) and 2.6 X 10(6) M-1 S-1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.

Pancreatic explants from Syrian hamsters were maintained in culture for determination of the fate of the zymogen granules in the acinar cells. By 6 hours of culture the zymogen granule matrix in many cells becomes nonhomogeneous, and electron-lucent spaces appear. Autoradiography performed 48 hours after pulse labeling shows that several of the altered zymogen granules contain radioactively labeled proteins. Hence these altered granules are mature zymogen granules undergoing regression. Ultrastructural analyses indicate that the zymogen granules are degraded intracellularly in the lysosomes by autophagy, by direct fusion between the lysosomes and the zymogen granules, as well as by engulfment of intact zymogen granules by the lysosomes. Budding of cytoplasm into zymogen granules is also frequently observed. Acid phosphatase histochemistry and anti-amylase immunohistochemistry were used for demonstration of presence of hydrolases and of secretory material in the degenerating zymogen granules. Cycloheximide and low temperature inhibit the degradation of zymogen granules and enhance the short-term viability of the explant. Secretagogues stimulate secretion but have little or no effect on the degradation of zymogen granules or on explant viability. The lysosomes participate in the intracellular degradation of zymogen granules in acinar cells of pancreatic explants. The induction of these lysosomal catabolic processes correlates with the structural alteration of pancreatic acinar cells. The intracellular degradation of secretory membranes and products by the lysosomes may play a role in the adaptation of pancreatic acinar cells to injury.

Auditory event related potentials and contingent negative variation (CNV) were recorded in a series of behavioral tasks from 27 hospitalized human volunteers before, early during (1--3 days), late during (10--14 days) and after chronic THC intoxication at two dose levels (70--90 mg/day or 180--210 mg/day in divided doses). Twelve additional hospitalized volunteers served as a control group. The N1 component of the auditory event related potential in all tasks was depressed when tested early during 180--210 mg/day THC administration. No N1 changes were seen for the subjects receiving 70--90 mg/day or the control subjects. Ten to 14 days during the chronic THC perioe CNV was depressed in all tasks except the control task for both doses at the early THC testing. Only at the most difficult task at the higher of the doses was the CNV depressed at the test late in the THC period. The changes seen in the N1 and the CNV may be indicative of different aspects of a common attentional alteration during stimulus processing. The results on our more difficult tasks suggest the alteration may continue in complex stimulus processing tasks in heavy marijuana users.

Amplitude changes in the auditory event related response reflect differences in linguistic content and the level of processing of spoken sentences. Thus, the neurophysiology of the cognitive processes underlying speech perception can be evaluated by noninvasive techniques. Semantically correct, semantically incorrect, and grammatically incorrect sentences were processed either semantically or syntactically by 25 subjects while their EEGs were recorded. The amplitude of the P250 component to each word varied with processing level. A slow positive wave appeared after semantically correct sentences regardless of the linguistic processing level. N480 and P780 components were observed following words which made the sentences incorrect.

To evaluate the electrophysiologic and neurohumoral effects of cocaine on the sinus node, atrium, atrioventricular node, and His-Purkinje system, 14 conscious dogs were studied before and after an infusion titrated to maximum tolerance or to increase the systolic blood pressure by at least 15%. Plasma cocaine and catecholamine levels, blood pressure, surface and intracardiac electrograms were recorded. Programmed electrical stimulation was performed from the right atrium. The sinoatrial conduction time, paced PR interval, atrioventricular nodal conduction time, atrioventricular nodal effective refractory period and blood pressure increased. Plasma noradrenaline, adrenaline and dopamine increased and remained at two to three times control levels throughout the study although plasma cocaine levels declined. Adrenaline levels were strongly correlated with the hemodynamic response while plasma cocaine levels were not. No sustained spontaneous atrial or ventricular arrhythmias were recorded after cocaine infusion. Atrial fibrillation with a slow ventricular response was induced by pacing after cocaine in only three dogs. There were no significant changes in pacing threshold, right intra-atrial conduction time, infranodal conduction time, heart rate, QRS or QT. No pacing induced infranodal block occurred. The cocaine dose rate infused was 2.8 +/- 1.2 mg/kg; cocaine plasma levels were 1402 +/- 885 ng/mL immediately after the initial infusion and 525 +/- 321 ng/mL at the end of the study. It is concluded that in normal canine heart cocaine increases blood pressure, sinoatrial conduction time, atrioventricular nodal effective refractory period, paced PR interval, atrioventricular nodal conduction time, plasma catecholamines and susceptibility to atrial fibrillation. These findings cannot be explained solely by increased sympathetic nervous system activity and suggest participation of the parasympathetic nervous system.

Pregnancy in female carriers of abnormal hemoglobins with great avidity for oxygen provides a unique opportunity to assess the importance of the usual difference in oxygen affinity between fetal and maternal blood. Outcome of pregnancy was recorded for carriers of hemoglobins Bethesda, Osler, and Yakima, whose p50s (9.5, 9.1, and 12 mm Hg at pH 7.4) were far lower than that of a normal fetus (23 mm Hg at pH 7.3). Neither spontaneous abortions nor intrauterine growth retardation could be attributed to the presence of high oxygen affinity in the mothers. In vitro simulations suggested that neither maternal or fetal polycythemia alone was sufficient to adjust for perturbation of the normal situation, and increased uterine and/or fetal blood flow probably provided additional compensation.

A method is described for extraction of the cocaine metabolite benzoylecgonine, conversion to the butyl ester derivative and gas chromatographic analysis using packed or capillary columns. Using a capillary column, cocaine and benzoylecgonine may be determined simultaneously. The extraction scheme has been designed to facilitate processing of large numbers of samples generated in pharmacokinetic studies. Structural analogues, m-toluylecgonine and m-toluylecgonine methyl ester, are used as internal standards. Concentrations as low as 10 ng/ml in 1-ml samples of plasma or urine are readily determined. Between-run coefficients of variation were 1.01% for cocaine and 4.18% for benzoylecgonine for concentrations of 75 and 350 ng/ml, respectively.

The effects of cocaine by two routes of administration were studied on the resting, awake human EEG during a 2 min sequential subtraction task. Fifty subjects were given 1 of 3 intravenous cocaine doses (0.2, 0.4 and 0.6 mg/kg). Thirty-three subjects received 1 of 3 oral doses of cocaine (2, 3 and 4 mg/kg). The EEG was analyzed as spectral power for delta, theta, alpha and beta bands. At each dose for both routes of administration, cocaine increased beta power. The increase was observed at the 5 min post-drug test session for the subjects given intravenous cocaine and at both 45 and 75 min test sessions for subjects given oral doses. In addition, a decrease in delta power was found at the 5 min test for the intravenous group and theta power was decreased at the 45 min test session. The increase in beta power was correlated with the area under the cocaine plasma versus time curve, but not with the cardiovascular effects of cocaine. The increased beta activity observed with cocaine may be a consequence of the direct stimulation of a central noradrenergic arousal system.

The effects of cocaine on electroencephalographic indices, information processing and motor performance were measured in 12 normal volunteers. They were tested with an auditory continuous performance task before, 60 and 180 min after a 3 mg/kg oral dose of cocaine hydrochloride or placebo in a double-blind counterbalanced crossover design. EEG from Fz, Cz and Pz were averaged for the eye movement-free correct trials. N100, P200, P300 components and CNV were measured. Cocaine increased N100 amplitude for the warning stimulus and CNV amplitude. P300 amplitude and latency, reaction time and sensory sensitivity (d') were not altered by cocaine. The increase in N100 and CNV amplitude may reflect mobilization of additional attentional resources to produce optimal performance on the task when subjects were distracted by the cardiovascular, subjective and other cocaine effects.

Hemolysates of erythrocytes from more than a quarter million people in Alabama were electrophoresed on cellulose acetate, pH 8.4, and those samples exhibiting an abnormality were also electrophoresed in citrate agar, pH 6.0. The globin chains of mutants other than Hb S and C were electrophoresed in urea-mercaptoethanol buffers at both pH 8.9 and pH 6.0, and 60 of them were also analyzed structurally. Of about 6000 samples from whites, only three contained abnormal hemoglobins--Hb D Los Angeles, Hb J Baltimore, and one unidentified. Of 249,000 samples from blacks, about 29,000 contained electrophoretically detectable abnormalities, most of them associated with Hb S or C, present in a frequency of about 9% and 3%, respectively. About 1000 samples resolved into patterns of potential clinical significance. Twenty other mutant hemoglobins were detected, in various genetic combinations in 164 kindreds; four of these-Hb Alabama, Montgomery, Titusville, and Mobile--were previously unknown. The methods used are rapid, economical, and well suited for large scale surveys. They provide highly specific characterizations of many mutant hemoglobins, and no discrepancies were found between the presumptive identifications based on these characterizations and the definitive identifications obtained from structural analyses.

Hemoglobin Ohio [beta 142 (H20) Ala replaced by Asp] was found in three members of a white family, all of whom showed erythrocytosis. The variant hemoglobin has a high oxygen affinity, a reduced Bohr effect, and diminished cooperativity. The functional abnormalities of Hb Ohio are explained by the proximity of the substituent beta 142 residue, both to beta 143 His, which is involved in the DPG binding site of hemoglobin, and to the critical C terminal region of the beta chain, which participates in the stabilization of the deoxy (T) conformation.

Sequence analysis of epsilon and gamma genes and encoded globins and high-pressure liquid chromatography analysis of globin compositions in blood hemolysates obtained from embryos, fetuses and adults show that the prosimian primate Galago crassicaudatus expresses its epsilon and gamma genes only embryonically. Since rabbit, mouse and galago all have embryonic gamma genes but simian primates have fetal gamma genes, we conclude that gamma E evolved into gamma F in stem-simians. An elevated non-synonymous substitution rate characterizes this transition. The alignment of epsilon and gamma nucleotide sequences and the parsimoniously reconstructed evolutionary history of these sequences identify several anciently conserved cis-regulatory elements (phylogenetic footprints) important for gamma expression in primates and also cis-mutations which may have been involved in the recruitment of the gamma gene to a fetal program in simian primates.