Characterization of the iron oxide phases formed during the synthesis of core-shell FexOy@C nanoparticles modified with Ag.

Core-shell FexOy@C nanoparticles (NPs) modified with Ag were studied with x-ray diffraction, transmission electron microscopy, energy dispersive elemental mapping, Mössbauer spectroscopy, static magnetic measurements, and optical magnetic circular dichroism (MCD). FexOy@C NPs synthesized by the pyrolysis process of the mixture of Fe(NO3)3 · 9H2O with oleylamine and oleic acid were added to a heated mixture of oleylamine and AgNO3 in different concentrations. The final product was a mixture of iron oxide crystalline NPs in an amorphous carbon shell and Ag crystalline NPs. The iron oxide NPs were presented by two magnetic phases with extremely close crystal structures: Fe3O4 and γ-Fe2O3. Ag is shown to form crystalline NPs located very close to the iron oxide NPs. An assumption is made about the formation of hybrid FexOy@C-Ag NPs. Correlations were obtained between the Ag concentration in the fabricated samples, their magnetic properties and the MCD spectrum shape. Introducing Ag led to a approximately linear decrease of the NPs saturation magnetization depending upon the Ag concentration, it also resulted into the MCD spectrum shift to the lower light wave energies. MCD was also studied for the Fe3O4@C NPs synthesized earlier with the same one-step process using different heat treatment temperatures, and MCD spectra were compared for two series of NPs. A possible contribution of the surface plasmon excitation in Ag NPs to the MCD spectrum of the FexOy@C-Ag NPs is discussed.

Rapid identification of Salmonella using Hektoen enteric agar and 16s ribosomal DNA probe-gold nanoparticle immunochromatography assay in clinical faecal specimens.

UNLABELLED: A rapid identification of Salmonella, one of the most common foodborne pathogens worldwide, in clinical patients can enable better rational managements and prevent further outbreaks. The traditional immunochromatography using antibody-gold nanoparticles (Ab-AuNPs), such as the home pregnancy test, has been used for the Salmonella detection. In this study, we developed a new and rapid method using DNA probe-AuNPs for the detection of 16s ribosomal DNA of Salmonella. To evaluate the proposed method in clinical specimens, we performed a clinical test by identifying 159 stool samples on Hektoen agar containing black or crystalloid colonies using the method and the VITEK 2 system for confirmation. Eighty of the isolates were correctly identified as Salmonella to achieve 100% sensitivity. Seventy-five samples were correctly identified as non-Salmonella spp., but four were incorrectly identified as Salmonella. The specificity was 94·93%. The assay time is about 30 min after the DNA purification. The time-consuming and labour-intense biochemical tests can be replaced. We demonstrated that this assay is a rapid, convenient and cost-effective tool for Salmonella identification of clinical faecal samples, which is worth for further promotion and clinical use. This is the first application of using 16s ribosomal DNA probe-Au-NPs and immunochromatography on clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first application of using 16s ribosomal DNA probe-gold nanoparticles and immunochromatography method on clinical samples with sensitivity 100% and specificity 94·93%. The assay time is about 30 min after the DNA purification. We find this assay a rapid, convenient, sensitive and inexpensive tool for Salmonella identification of clinical faecal samples, which is worth further promotion and clinical use and can replace the traditional time-consuming and labour-intense biochemical tests. The potential benefit of this approach is to develop a rapid point-of-care test that provides results while the patient is still at the doctors' office.

A study of PVRL1 mutations for non-syndromic cleft lip and/or palate among Taiwanese patients.

Mutations of codons 185 and 323, especially the W185X mutation, of the PVRL1 gene among non-syndromic cleft lip and/or palate (CL/P) patients and normal controls in Taiwan were studied in order to determine whether there are mutations that play a part in the formation of non-syndromic CL/P. A total of 76 patients were enrolled; 66 sporadic non-syndromic CL/P patients and 10 normal controls. The mutation survey for codons 185 and 323 was conducted using a polymerase chain reaction and DNA sequencing. Neither mutations of codons 185 and 323 were noted for any of the 76 patients (152 alleles), nor were found any other mutations in either exon 3 or 5 of the PVRL1 gene. These results suggest that mutations of the PVRL1 gene at codons 185 and 323, especially the W185X mutation, do not participate in the formation of CL/P within the Taiwanese population examined.

Reduced haloperidol and haloperidol: effects on homovanillic acid in caudate and prefrontal cortex.

The effects of acute administration of reduced haloperidol (RHAL) on homovanillic acid (HVA) in the caudate and prefrontal cortex were examined in rats. Haloperidol (HAL) was used as a reference compound. Concentrations of HVA and HAL were measured by HPLC/ECD. The maximal HVA response time was 3 hr after the injection, in both caudate and prefrontal cortex, for both RHAL and HAL. The potency of RHAL in the elevations of HVA in the caudate and prefrontal cortex was only about one-third to one-fifth that of HAL. The concentrations of HAL in the prefrontal cortex and caudate after RHAL administration were just about one-third to one-fifth those after HAL administration. These results suggest that less antidopaminergic activity of RHAL in this neuroleptic test might be explained by the lesser conversion of RHAL to HAL.

Acute and chronic effects of haloperidol on plasma and brain homovanillic acid in the rat.

The effects of acute and chronic administration of haloperidol on homovanillic acid (HVA) in plasma and the brain were examined in the rat. Acute haloperidol treatment (1 mg/kg) resulted in highly significant elevations in HVA within 30 min and produced a maximal increase of HVA in 3-6 hr in both plasma and the whole brain. The response of brain HVA to increasing doses (0.05-30 mg/kg) of haloperidol showed an inverted U pattern. Plasma HVA showed a very flat response to lower doses (less than or equal to 5 mg/kg) of haloperidol and a dramatically elevated one to higher doses (greater than or equal to 10 mg/kg). Haloperidol produced a parallel increase in plasma and brain HVA at lower doses (less than or equal to 2 mg/kg) only. After chronic administration of haloperidol for up to 28 days, the response of HVA in plasma correlated mainly with, but tolerated later than, those in the whole brain and the olfactory tubercle.

Human placental norepinephrine transporter mRNA: expression and correlation with fetal condition at birth.

The purpose of this study was to determine the primary form of human placental norepinephrine transporter (hNET) mRNA expressed in the human placenta and to compare the level of expression in normal pregnancies and in pregnancies complicated by drug exposure or other forms of physiological derangement. We used the hNET cDNA to measure RNA extracted from placenta and examined placental RNA following complicated and uncomplicated pregnancies. To compare transporter expression and its relation to fetal condition at birth, umbilical arterial plasma catecholamine levels, umbilical arterial blood gases and placental transporter mRNA level were compared by linear regression analysis. Uncomplicated pregnancies had a higher level of placental norepinephrine transporter mRNA than complicated pregnancies. An inverse relationship between umbilical cord norepinephrine level and transporter expression was demonstrated. We conclude that placental transporter expression represents an important and newly described metabolic function of the placenta. Placental catecholamine clearance mediated via the placental NET may be important in the pathophysiology of disorders associated with placental dysfunction, impaired placental blood flow or intrauterine growth retardation. This may also explain the adverse effects of drugs, such as cocaine, which block catecholamine transport.

Placental biogenic amine transporters: in vivo function, regulation and pathobiological significance.

The biogenic amine transporters are part of a large family of plasma membrane transporters. These carriers mediate the re-uptake of neurotransmitters from the synaptic cleft and plasma compartments. Re-uptake process is inhibited by drugs like cocaine, fluoxetine and tricyclic antidepressants. There are specific transporters for norepinephrine, epinephrine, dopamine and serotonin. The placenta expresses the norepinephrine and serotonin transporters, which is unusual as they are otherwise expressed predominantly in neuronal tissue. Fetal catecholamine clearance rate is higher than under any other physiological conditions and is mediated in large measure by the placental transporters. The high intrauterine catecholamine secretion and clearance rates are part of the unique fetal neuroendocrine milieu. They condition the fetus to a high capacity for catecholamine secretion in the early postnatal period when elevated sympathoadrenal system activity is vital for postnatal survival. Because of the prominent catecholamine clearance rate, the fetus is vulnerable to the adverse effects of re-uptake inhibitors. Understanding the mechanisms of expression and regulation of placental biogenic amine transporters is important to the pathobiology of fetal conditions associated with elevated catecholamine levels or intrauterine exposure to uptake inhibitors like cocaine.

A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the beta1-adrenergic receptor gene.

The effects of glucocorticoids on expression of the beta1-adrenergic receptor (beta1AR) gene have been varied. To study the mechanism underling hormonal regulation of the beta1AR, transient transfection of progressively deleted ovine beta1AR promoter fragments was used to identify a 43-bp region (-1274 to -1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) and confers glucocorticoid responsiveness. Using DNase I footprinting and electrophoretic mobility shift assays (EMSA), we demonstrated the GRU was composed of a palindrome, 5'-TAATTA-3', which is a core binding motif for the homeodomain proteins, an E-box (5'-CACGTG-3'), binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element (GRE) half-site (5'-TGTTCT-3'). EMSA demonstrated that the GRE half-site is critical for GRU-protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Co-transfection of a plasmid expressing a c-myc antisense construct significantly reduced glucocorticoid responsiveness of the ovine beta1AR promoter. Furthermore, expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic, reduced in newborn and not detectable in adult heart. We conclude that the ovine beta1AR promoter contains a novel, functional GRU and that glucocorticoid receptor (GR) and the Myc/Max family proteins are involved in the cell-specific nuclear factor binding and transactivation via this element. The results suggest an alternative pathway through which glucocorticoids may exert their effects on genes lacking a full consensus GRE.

Effects of gender and age on plasma levels of clozapine and its metabolites: analyzed by critical statistics.

BACKGROUND: Previous reports concerning the effects of gender and age on steady-state plasma concentrations of clozapine and its major metabolites, norclozapine and clozapine-N-oxide, have been controversial. Since the frequency distribution of the plasma levels is asymmetrical and skewed to the right, the statistical methods (such as analysis of variance and regression analysis) used earlier are actually inappropriate for analyzing the effects of the variables on the concentrations and might contribute to the inconsistent results. The goal of the present study, with befitting statistics, is to measure the potential effect of dose, gender, age, and body weight on plasma levels of clozapine and its 2 major metabolites. METHOD: We retrospectively analyzed data from a therapeutic drug monitoring study for steady-state plasma clozapine, norclozapine, and clozapine-N-oxide levels that was conducted in a large group of Chinese schizophrenic inpatients (male:female ratio = 83:79; age range, 33.8 +/- 9.3 years). The daily doses of clozapine had ranged from 100 to 900 mg, with a mean +/- SD value of 379.5 +/- 142.2 mg. Plasma concentrations had been measured using high-performance liquid chromatography with ultraviolet detection. Multiple linear regression was adopted to quantify the effects of various factors on the plasma levels. The natural logarithm of the plasma level was used as the dependent variable to overcome the skewness problem. RESULTS: After adjusting the effects of gender, age, and body weight by multiple linear regression, each 1-mg increment in the daily dose could raise the clozapine level by 0.31%, norclozapine by 0.27%, and clozapine-N-oxide by 0.16%. Female patients had 34.9% higher clozapine levels and 36.3% higher norclozapine, with other variables being controlled. No sex differences were demonstrated for clozapine-N-oxide levels. Each 1-year increment in age would elevate the clozapine level by 1.1%, norclozapine by 1.0%, and clozapine-N-oxide by 1.0%. Body weight could not influence the levels of these compounds. CONCLUSION: The present results suggest that women possess higher plasma levels (about one third higher) of clozapine and norclozapine, but not the N-oxide metabolite. Each addition of 1 year in age elevated clozapine and either metabolite's levels by about 1%. Furthermore, every 1-mg increase in the daily dose raised clozapine and norclozapine concentrations by approximately 0.3%. These findings could assist clinicians in optimizing clozapine dosing strategies.

Cloning and sequence analysis of the rat norepinephrine transporter promoter.

We isolated a 2.5-kb fragment of the promoter for the rat norepinephrine transporter (NET) gene. The transcription start site was identified approximately 200 base pairs upstream from the translation start site. Several potential regulatory elements were identified by sequence analysis. The structure of the rat NET promoter was compared to mouse and human. Expression studies in placental and neuroblastoma cells suggested the presence of a 'repressor' element more than 500 base pairs upstream from the transcription start site. These studies provide the basis for examination of transcriptional regulation of this gene and for understanding its temporal and tissue-specific modes of regulation.

Placental biogenic amine transporters: cloning and expression.

During intrauterine development, catecholamine turnover (production and clearance rates) is higher than under any other circumstances. This is mediated in large part by placental clearance of circulating catecholamines via a cocaine-sensitive, neuronal transporter-dependent mechanism. In order to confirm the molecular mechanisms for placental transport, we screened an ovine placental cDNA library for biogenic amine transporters. We report here the identification of two biogenic amine transporters with sequences very similar to their neuronal counterparts. One is an ovine serotonin transporter (oSERT) with > 90% homology to the human neuronal SERT. Expression studies confirm transport and competitive binding affinities consistent with a SERT transporter. We have also isolated a partial sequence for the ovine norepinephrine transporter (oNET). These results confirm the placental expression of plasma membrane biogenic amine transporters. We suggest the exaggerated fetal vulnerability to uptake inhibitors, like cocaine, may be due to blockade of placental biogenic amine transport.

Region-specific changes of GABAA receptors by tolerance to and dependence upon pentobarbital.

An experimental model for inducing tolerance to and dependence upon pentobarbital was characterized. Rats were infused with pentobarbital, 300 micrograms/10 microliter per h i.c.v. for 6 or 7 days, through pre-implanted cannulas by osmotic minipumps. Measurement of brain and serum levels showed that pentobarbital remained mainly inside of the brain. Measurements of sleeping time and susceptibility to convulsant-induced seizures indicated a substantial degree of tolerance and dependence (24 h after termination of infusion). Results of GABAA receptor binding assays showed marked regional variations. While [35S]t-butylbicyclophosphorothionate (TBPS) binding sites were increased in frontal cortices and striata of pentobarbital-dependent animals, KD was increased in striata of tolerant animals. Dependence upon pentobarbital was correlated with increased [3H]flunitrazepam binding sites in all three regions examined. Both high and low affinity [3H]muscimol binding sites were increased in dependent animals, but low affinity sites were decreased in frontal cortices of tolerant animals. KDs of [3H]muscimol high affinity sites were increased in cerebellum after animals developed dependence upon pentobarbital. KDs of [3H]muscimol low affinity sites were decreased in striata of pentobarbital tolerant animals. These findings further support the hypothesis that GABAA receptors in discrete areas of the brain have different subunit compositions and are regulated differentially by pharmacological modulators.

Regulation of beta 1-adrenoceptors by glucocorticoids and thyroid hormones in fetal sheep.

The effects of betamethasone alone or in combination with thyroxine (T4) on ovine fetal beta-adrenoceptors were investigated at the molecular level. Ovine fetuses (126 days gestation; term = 150 days) were treated with a single ultrasound-guided intramuscular injection of 0.5 mg/kg betamethasone, betamethasone + 50 micrograms/kg T4, or saline. Forty-eight h after injection, lambs were delivered by cesarean section and evaluated three h for postnatal adaptation. Myocardial beta-adrenoceptor equilibrium dissociation constant (Kd) and maximal receptor density (Bmax), as assessed by [3H]dihydroalprenolol binding, were not significantly different in drug-treated groups compared to the control group. Northern hybridization and RNase protection assays of myocardial total RNA probed with a sheep beta 1-adrenoceptor riboprobe confirmed no changes in expression at the level of the gene. Levels of beta 1-adrenoceptor mRNA in the lung and brain were also unaffected by the treatments. Because other genes are responsive to glucocorticoids and thyroid hormones at this stage, the absence of up-regulation of beta-adrenoceptor number and steady-state levels of mRNA coding for beta 1-adrenoceptor following fetal corticosteroid and thyroid hormone treatment may indicate a specific, developmentally regulated repressor mechanism.

Effects of cocaine on tyrosine hydroxylase activity in brain areas from SHR and WKY.

Basal tyrosine hydroxylase activity was the same in the nuclei accumbens and hypothalami of WKY and SHR. Basal striatal enzyme activity was lower in SHR than in WKY. Acute and subacute cocaine administration altered enzyme activity only in striata and nuclei accumbens of WKY. The central dopaminergic system of SHR appears to be less active and less sensitive to cocaine than that of WKY.

Differential effects on GABAA receptor gamma 2-subunit messenger RNA by tolerance to and withdrawal from pentobarbital--an in situ hybridization study.

The heterogeneity of the GABAA receptors has been confirmed structurally and functionally. The present study demonstrates the pharmacological heterogeneity of the GABAA receptors. Rats were rendered tolerant to pentobarbital by continuous intracerebroventricular infusion via osmotic minipumps and abruptly withdrawn from pentobarbital. In situ hybridization of mRNA coding for the GABAA receptor gamma 2-subunit showed decreases of mRNA levels in superior and inferior colliculus in pentobarbital tolerant rats compared to rats in withdrawal. In rats 24-hr after withdrawal from pentobarbital, increases of mRNA levels in neocortex, piriform cortex and in granular and Purkinje cell layers of the cerebellum were observed. These results indicate the fast adaptation of GABA synapses in response to abrupt withdrawal from chronic pentobarbital treatment. The differential responsiveness seen in different areas further confirms the pharmacological heterogeneity of the GABAA receptors. The observed increases and decreases of mRNA may underlie, at least in part, the previously reported changes in Bmax of GABAA receptor ligand binding sites.

Ketone reductase activity and reduced haloperidol/haloperidol ratios in haloperidol-treated schizophrenic patients.

Twenty schizophrenic patients were treated with a fixed haloperidol (HL) dose of 20 mg/day for 4 weeks. The conversion of HL to its reduced metabolite (reduced haloperidol, RH) occurs via the ketone reductase enzyme. RH is also converted back to HL by the cytochrome P450 2D6 isozyme. Ketone reductase activity can be measured in red blood cells. Plasma HL and RH levels were assayed by high performance liquid chromatography. Blood samples were obtained at baseline and during weeks 2 and 4 of HL therapy. Seventeen of 20 patients had ketone reductase values < 3. A significant correlation between ketone reductase and RH/HL plasma ratios was observed at week 4 in these 17 patients. Patients with ketone reductase activity < 3 could represent a subgroup of patients that metabolize HL differently. The wide interpatient variability observed with HL and RH plasma levels in HL-treated patients could reflect differences in ketone reductase activity and the metabolic status of debrisoquin hydroxylase (cytochrome P450IID6) in psychiatric patients.

Tolerance development to butorphanol: comparison with morphine.

In order to evaluate and to compare the time course, dose response, and the degree of tolerance development to butorphanol and morphine, rats were continuously intracerebroventricularly (ICV) infused with saline vehicle (1 microliter/h), butorphanol (6.5, 13, 26, and 52 nmol/microliters/h), or morphine (1.6, 6.5, and 26 nmol/microliters/h) through osmotic minipumps for 1 to 3 days. The tail-flick responses were determined pre-, during, and postinfusion. Tolerance to morphine developed faster than that to butorphanol. The antinociceptive response to the ICV challenge dose (6 h after the termination of drug infusion) of butorphanol or morphine was decreased significantly and there was a negative correlation between the dose of the drug infused and the observed antinociceptive response. In terms of butorphanol and morphine tolerance, a parallel rightward shift in the dose-response curve was produced with the degree of shift proportional to the log of the infusion dose. In tail-flick tests, the shifts of the dose-response curves for butorphanol and morphine in tolerant animals were 11.8- and 46.3-fold, respectively. However, in the acetic acid writhing test, the shifts of the dose-response curves for butorphanol and morphine in tolerant animals were 11.3- and 11.7-fold, respectively. These results suggest that there is a greater degree of tolerance to morphine than there is to butorphanol, but the degree of butorphanol tolerance is still substantial. In addition, two pain assays (tail flick vs. writhing) yielded different estimations of tolerance in a comparison of morphine and butorphanol.

Crosstolerance between butorphanol and morphine in rats.

To investigate the antinociceptive effects of morphine and U-50,488 after continuous administration with butorphanol, rats were intracerebroventricularly (ICV) infused with butorphanol (26 nmol/microliters/h) through osmotic minipumps for 3 days. Six hours after termination of infusion, the rats were challenged with different doses of morphine or U-50,488. Antinociceptive effects, as assessed by tail-flick and acetic acid writing tests, were measured 15 min after challenge. Development of crosstolerance to morphine was evident in butorphanol-infused animals. The study also revealed that crosstolerance to butorphanol developed in continuously ICV morphine-infused animals. Continuous ICV infusion with butorphanol produced a marked rightward shift of the antinociceptive dose-response curve resulting from U-50,488 challenge. These results showed that there is an antinociceptive crosstolerance between butorphanol and morphine, and crosstolerance to U-50,488 developed in continuously butorphanol-infused animals. The present data suggested that chronic ICV treatment with high doses of butorphanol can lead to desensitization of the antinociceptive systems mediated through the central kappa as well as mu receptors in rats.

Esophageal reconstruction for hypopharyngoesophageal strictures after corrosive injury.

OBJECTIVE: To evaluate the surgical outcome of patients with caustic stricture of the hypopharyngoesophagus. MATERIALS AND METHODS: During a 25-year period, we performed esophageal reconstruction in 152 patients with diffuse or multiple caustic esophageal stricture. Of them, esophageal substitute was pulled up and anastomosed to the hypopharynx in 50 (33%) patients, and anastomosed to the cervical esophagus in the other 102 (67%) patients. Patients whose esophageal substitute anastomosed to the hypopharynx were enrolled to the present study. Among these 50 study patients, 13 underwent ablation of damaged organs and feeding jejunostomy in acute stage of corrosive injury, and the remaining 37 patients were initially organ preserved with or without feeding gastrostomy or jejunostomy. Six patients had respiratory distress caused by laryngotracheal stricture. The ileocolon (28/50) was commonly used as an esophageal substitute in reconstruction and most substitutes (43/50) went through the substernal route. RESULTS: There was one operative death. Eight (16%) patients had major early postoperative complications. Six patients underwent revision for late stenosis of hypopharyngeal anastomosis, and one redoing reconstruction using the jejunum because of failure of the transplanted ileocolon. Postoperatively, swallow function and maintaining body weight were considered good in 42 patients (84%) after an average of 8 months follow-up. Five of six patients who underwent concomitant tracheostomy or laryngosurgery for laryngotracheal stricture got unsatisfactory result. The surgical outcome of the study patients was worse than that in patients with esophageal substitute anastomosed to a healthy cervical esophagus. In the later group of patients, 95/102 (93%) had good swallow function and only 7/102 (6.8%) had major early complications. CONCLUSION: Caustic stricture of the hypopharyngoesophagus is a challenging reconstructive problem. A successful reconstruction requires a correct hypopharyngeal opening and anastomosis, a good esophageal substitute, and a patent esophageal route and airway.

A cloning strategy for G-protein-coupled hormone receptors: the ovine beta 1-adrenergic receptor.

Regulation of beta 1-adrenergic receptors is unusual in developing animals. For example, glucocorticoid-and thyroid hormone-responsiveness for several genes is seen in animals treated during fetal life but beta 1-responsiveness is not seen until after birth. In order to investigate this at the transcriptional level, the ovine beta 1 receptor gene was cloned from a sheep genomic library. An approach using high-stringency screening with cDNA probes and oligonucleotides from regions of human and rat genes conserved but unique to the beta 1 receptor but not to other seven transmembrane, G-protein-coupled receptors. Over 800,000 clones were screened from which 40-50 positive clones were identified by each of the probes. There was, however, only a single clone which was recognized by each of the probes. A 5-kb insert was subcloned and shown to contain sequences which hybridized to each of the probes. Using the restriction map of the rat beta 1 receptor, a 1.0-kb Pst1 internal fragment was further subcloned for sequence identification. Confirmation of this fragment as the ovine beta 1 receptor was based on homology of the beta 1 receptor from other species and tissue distribution of mRNA. Nucleotide sequence homology was 93% with the human beta 1 receptor and 84% with rat. Amino acid sequence homology was > 75% and approached 100% in the transmembrane regions. The approach described represents a practical approach to cloning and identification of hormone receptors from the highly homologous members of the seven-transmembrane, G-protein-coupled receptors.