The experience of working on a locked acute psychiatric ward.

This study's aim was to elucidate health-care staff experience of working on a locked acute psychiatric ward. In many countries changes in health care has contributed to fewer beds available in inpatient care, and a concentration of patients with severe psychiatric conditions. This implies a changing work environment in acute psychiatric care. Qualitative interviews with health-care staff (n= 10) were carried out on a ward for patients with affective disorder and eating disorder in a Swedish hospital. Qualitative content analysis was used. Four themes were identified from the data: 'undergoing changes in care delivery', 'feeling a need for security and control', 'managing the demands at work' and 'feeling a sense of responsibility'. This study adds to earlier research into how a sense of responsibility can place a significant burden on health-care staff working on a locked psychiatric ward and also contribute to increased control of patients. This study also shows that relationships and power structures among health-care staff need to be addressed when organizational changes are made in care delivery. Further research is needed to reach a comprehensive understanding of care on locked acute psychiatric wards, including a development of nursing and medicine as knowledge domains in one common context.

Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons.

Some women have negative mood symptoms, caused by progestagens in hormonal contraceptives or sequential hormone therapy or by progesterone in the luteal phase of the menstrual cycle, which may be attributed to metabolites acting on the GABA-A receptor. The GABA system is the major inhibitory system in the adult CNS and most positive modulators of the GABA-A receptor (benzodiazepines, barbiturates, alcohol, GABA steroids), induce inhibitory (e.g. anesthetic, sedative, anticonvulsant, anxiolytic) effects. However, some individuals have adverse effects (seizures, increased pain, anxiety, irritability, aggression) upon exposure. Positive GABA-A receptor modulators induce strong paradoxical effects including negative mood in 3%-8% of those exposed, while up to 25% have moderate symptoms. The effect is biphasic: low concentrations induce an adverse anxiogenic effect while higher concentrations decrease this effect and show inhibitory, calming properties. The prevalence of premenstrual dysphoric disorder (PMDD) is also 3%-8% among women in fertile ages, and up to 25% have more moderate symptoms of premenstrual syndrome (PMS). Patients with PMDD have severe luteal phase-related symptoms and show changes in GABA-A receptor sensitivity and GABA concentrations. Findings suggest that negative mood symptoms in women with PMDD are caused by the paradoxical effect of allopregnanolone mediated via the GABA-A receptor, which may be explained by one or more of three hypotheses regarding the paradoxical effect of GABA steroids on behavior: (1) under certain conditions, such as puberty, the relative fraction of certain GABA-A receptor subtypes may be altered, and at those subtypes the GABA steroids may act as negative modulators in contrast to their usual role as positive modulators; (2) in certain brain areas of vulnerable women the transmembrane Cl(-) gradient may be altered by factors such as estrogens that favor excitability; (3) inhibition of inhibitory neurons may promote disinhibition, and hence excitability. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

Encounters in a locked psychiatric ward environment.

This focused ethnographic study aims at describing encounters in the healthcare environment on a locked psychiatric ward. It was carried out in Sweden on an acute psychiatric ward for patients with affective and eating disorders. Data were collected through participant observations and informal interviews, and analysed by qualitative content analysis. The result shows that the healthcare environment on this locked psychiatric ward offered a space for encounters between people, in a continuum from professional care to private meetings and social events. It included joy and friendship as well as unintentional insights into other patients' suffering. The characteristics of the encounters formed three themes: the caring relationship, the uncaring relationship and the unrecognized relationship. The caring and the uncaring relationship concerned relationships between staff and patients or their next of kin. These revealed contrasting qualities such as respect and flexibility as well as lack of respect and mistrust. The unrecognized relationship theme visualized the patients' relationships with each other and included both supportive and intrusive elements that were probably significant for the outcome of care. The unrecognized relationship contributes with new knowledge about conditions for patients in inpatient care, and indicates that the patients' relationships with each other merit greater attention.

GABA-site antagonism and pentobarbital actions do not depend on the alpha-subunit type in the recombinant rat GABA receptor.

AIM: The roles of alpha-subunits on the gamma-aminobutyric acid (GABA)-site antagonism and pentobarbital actions were examined in rat recombinant GABA(A) receptors in Xenopus oocytes. METHODS: Experiments were performed with binary and ternary GABA(A) receptors containing alpha1-, alpha4- or alpha5-subunit by the two-electrode voltage-clamp technique. RESULTS: The potency of GABA was significantly higher in the alpha1beta2, alpha4beta2 and alpha5beta2 receptors compared with the alpha1beta2gamma2L, alpha4beta2gamma2L and alpha5beta2gamma2L receptors. However, the alpha5beta2 receptor possessed significantly lower GABA efficacy compared with the alpha5beta2gamma2L receptor. While the gamma2-subunit was essential to the potency of GABA, its influence on the apparent GABA-site antagonism was less profound. The antagonist affinity constants (K(B)) of bicuculline inhibition and slopes of Schild plots were similar between all types of ternary and binary receptors except alpha5beta2 receptor which was not tested. The pK(B)s and IC(50)s of the GABA-site antagonism were not significantly different between the alpha1beta2gamma2L, alpha4beta2gamma2L and alpha5beta2gamma2L receptors. Bicuculline blocked pentobarbital-activated currents in a reversible and non-competitive manner with the alpha1beta2gamma2L, alpha4beta2gamma2L, and alpha5beta2gamma2L receptors, indicating an allosteric inhibition of the GABA-site. No significant difference of bicuculline potencies in inhibiting GABA- and pentobarbital-activated currents was found between the alpha1beta2gamma2L, alpha4beta2gamma2L and alpha5beta2gamma2L receptors. CONCLUSION: The GABA-site antagonism does not depend on the subtype of alpha-subunits. Similarly, pentobarbital activates ternary receptors composed of different alpha-subunits in a bicuculline-sensitive manner. The potencies of bicuculline to inhibit pentobarbital-activated currents are identical with receptors containing alpha1, alpha4 or alpha5-subunit. The alpha1beta2 and alpha4beta2 receptors possess higher GABA potencies compared with the alpha1beta2gamma2L and alpha4beta2gamma2L receptors.

Tolerance development to Morris water maze test impairments induced by acute allopregnanolone.

The progesterone metabolite allopregnanolone, like benzodiazepines, reduces learning and impairs memory in rats. Both substances act as GABA agonists at the GABA-A receptor and impair the performance in the Morris water maze test. Women are during the menstrual cycle, pregnancy, and during hormone replacement therapy exposed to allopregnanolone or allopregnanolone-like substances for extended periods. Long-term benzodiazepine treatment can cause tolerance against benzodiazepine-induced learning impairments. In this study we evaluated whether a corresponding allopregnanolone tolerance develops in rats. Adult male Wistar rats were pretreated for 3 days with i.v. allopregnanolone injections (2 mg/kg) one or two times a day, or for 7 days with allopregnanolone injections 20 mg/kg intraperitoneally, twice a day. Thereafter the rats were tested in the Morris water maze for 5 days and compared with relevant controls. Rats pretreated with allopregnanolone twice a day had decreased escape latency, path length and thigmotaxis compared with the acute allopregnanolone group that was pretreated with vehicle. Pretreatment for 7 days resulted in learning of the platform position. However, the memory of the platform position was in these tolerant rats not as strong as in controls only given vehicle. Allopregnanolone treatment was therefore seen to induce a partial tolerance against acute allopregnanolone effects in the Morris water maze.

Effects of postischemic environment on transcription factor and serotonin receptor expression after permanent focal cortical ischemia in rats.

Housing rats in an enriched environment improves functional outcome after ischemic stroke, this may reflect neuronal plasticity in brain regions outside the lesion. Which components of the enriched environment that are of greatest importance for recovery after brain ischemia is uncertain. We have previously found that enriched environment and social interaction alone both improve functional recovery after focal cerebral ischemia, compared with isolated housing with voluntary wheel-running. In this study, the aim was to separate components of the enriched environment and investigate the effects on some potential mediators of improved functional recovery; such as the inducible transcription factors nerve growth factor-induced gene A (NGFI-A) and NGFI-B, and the glucocorticoid and serotonin systems. After permanent middle cerebral artery occlusion, rats were divided into four groups: individually housed with no equipment (deprived group), individually housed with free access to a running wheel (running group), housed together in a large cage with no equipment (social group) or in a large cage furnished with exchangeable bars, chains and other objects (enriched group). mRNA expression of inducible transcription factors, serotonin and glucocorticoid receptors was determined with in situ hybridisation 1 month after cerebral ischemia. Rats housed in enriched or social environments showed significantly higher mRNA expression of NGFI-A and NGFI-B in cortical regions outside the lesion and in the CA1 (cornu ammonis region of the hippocampus), compared with isolated rats with or without a running wheel. NGFI-A and NGFI-B mRNA expression in cortex and in CA1 was significantly correlated to functional outcome. 5-Hydroxytryptamine receptor 1A (5-HT(1A)) mRNA expression and binding, as well as 5-HT(2A) receptor mRNA expression were decreased in the hippocampus (CA4 region) of the running wheel rats. Mineralocorticoid receptor gene expression was increased in the dentate gyrus amongst wheel-running rats. No group differences were found in plasma corticosterone levels or mRNA levels of glucocorticoid receptor, corticotropin-releasing hormone, 5-HT(2C) or c-fos. In conclusion, we have found that social interaction is a major component of the enriched environment regarding the effects on NGFI-A and NGFI-B expression. These transcription factors may be important mediators of improved functional recovery after brain infarctions, induced by environmental enrichment.

Patients' experience of involuntary psychiatric care: good opportunities and great losses.

Patients who are involuntary admitted to psychiatric care are extremely vulnerable as a consequence of the control from others, and of the personal limitations due to a psychiatric disease that can influence their own control of their lives. This group of patients are seldom asked about their experiences of being cared for. In this study five involuntary hospitalized psychiatric patients narrated their experience of being subjected to involuntary psychiatric care. The aim of the study was to obtain a deeper understanding of this experience. The interview text was analysed by means of a phenomenological hermeneutic method. The result of the analysis gave a complex picture of both support and violation. On the one hand experiences of not being seen or heard, of loss of liberty and of violation of integrity were found. On the other hand, there were experiences of respect and caring and opportunities to take responsibility for oneself were offered. Being treated involuntarily in psychiatric care was interpreted as a balancing act between good opportunities and great losses.

Neuroactive steroids and central nervous system disorders.

Steroid hormones are vital for the cell life and affect a number of neuroendocrine and behavioral functions. In contrast to their endocrine actions, certain steroids have been shown to rapidly alter brain excitability and to produce behavioral effects within seconds to minutes. In this article we direct attention to this issue of neuroactive steroids by outlining several aspects of current interest in the field of steroid research. Recent advances in the neurobiology of neuroactive are described along with the impact of advances on drug design for central nervous system (CNS) disorders provoked by neuroactive steriods. The theme was selected in association with the clinical aspects and therapeutical potentials of the neuroactive steroids in CNS disorders. A wide range of topics relating to the neuroactive steroids are outlined, including steroid concentrations in the brain, premenstrual syndrome, estrogen and Alzheimer's disease, side effects of oral contraceptives, mental disorder in menopause, hormone replacement therapy, Catamenial epilepsy, and neuractive steroids in epilepsy treatment.

Serotonin 5-HT(1A) receptor mRNA expression in dorsal hippocampus and raphe nuclei after gonadal hormone manipulation in female rats.

Female ovarian steroids influence mood and cognition, an effect presumably mediated by the serotonergic system. A key receptor in this interplay may be the 5-HT(1A) receptor subtype. We gave adult ovariectomized female rats subcutaneous pellets containing different dosages of 17 beta-estradiol alone or in combination with progesterone, or placebo pellets, for 2 weeks. 5-HT(1A) receptor mRNA levels were analyzed by in situ hybridization in the dorsal hippocampus, dorsal and median raphe nuclei, and entorhinal cortex. Estradiol treatment alone reduced 5-HT(1A) gene expression in the dentate gyrus and the CA2 region (17 and 19% decrease, respectively). Estradiol combined with progesterone supplementation increased 5-HT(1A) gene expression versus placebo in the CA1 and CA2 subregions of the dorsal hippocampus (16 and 30% increase, respectively). Concomitantly, 5-HT(1A) mRNA expression was decreased by 13% in the ventrolateral part of the dorsal raphe nuclei, while no changes were found in the median raphe nucleus and entorhinal cortex. Chronic effects of ovarian hormones on 5-HT(1A) receptor mRNA expression appear tissue-specific and involve hippocampal subregions and the raphe nuclei. Modulation of 5-HT(1A) receptor gene expression may be of importance for gonadal steroid effects on mood and cognition.

Early and delayed induction of immediate early gene expression in a novel focal cerebral ischemia model in the rat.

This study aimed at evaluating changes in expression of immediate early genes in a new photothrombotic focal ischemia model that exhibits late spontaneous reperfusion and morphological restoration in the region-at-risk within the cerebral cortex. Gene expression was studied with Northern blots, in situ hybridization and immunohistochemistry. At early time points (1-4 h), nerve growth factor-induced gene A and B, and c-fos mRNAs, were quickly induced throughout the ipsilateral cortex, with no obvious differences between the region-at-risk and remote cortical areas. High concentrations of nerve growth factor-induced gene A and c-Fos proteins were present within the region-at-risk even when cortical cerebral blood flow was as low as 40% of control values. At 4 h the nerve growth factor-induced gene A mRNA and protein expression was significantly decreased in the hippocampus vs. naive controls. However, a small decrease was also found in sham-operated and anaesthetized controls. A late induction, at 5 days, of c-fos and nerve growth factor-induced gene B mRNAs was seen bilaterally in the hippocampus and also, in the case of nerve growth factor induced-gene B, in the contralateral cortex. A complex pattern of changes in immediate early gene expression occurs after reversible focal cortical ischemia. This may be important for tissue recovery as well as neuropsychiatric symptoms after stroke.

Selective effects on NGFI-A, MR, GR and NGFI-B hippocampal mRNA expression after chronic treatment with different subclasses of antidepressants in the rat.

There is a latency period of several weeks before the onset of clinical effect of antidepressant drugs. The detailed mechanisms underlying drug-induced adaptive neuronal changes are not known. To elucidate the involvement of changes in gene expression of candidate transcription factors, we treated rats for 21 days with buspirone, fluoxetine, 8-OH-DPAT and moclobemide. In situ hybridization was used to study mRNAs encoding NGFI-A, NGFI-B and the glucocorticoid receptors, MR and GR. NGFI-A mRNA expression increased profoundly in the hippocampal formation and the cerebral cortex after all drug treatments, especially after moclobemide treatment (77-122% increase); with the exception of buspirone. MR mRNA expression was induced in hippocampal CA1/CA2 subregions (27-37%) by all antidepressants, while moclobemide and 8-OH-DPAT significantly increased GR gene expression mainly in the CA1 region (31-44%). NGFI-B mRNA was significantly decreased in the hippocampal CA3 subfield (23%) and restrosplenial granular cortex (38%) by moclobemide treatment. There are selective effects of antidepressant drugs on specific transcription factors. These may be important for adaptive neuronal and neuroendocrine changes after antidepressant treatment including HPA axis negative feedback regulation.

Environmental enrichment alters nerve growth factor-induced gene A and glucocorticoid receptor messenger RNA expression after middle cerebral artery occlusion in rats.

Housing rats in an enriched environment after focal brain ischemia improves functional outcome without changes in infarct volume, suggesting neuroplastic changes outside the lesion. In this study, permanent occlusion of the middle cerebral artery was followed by housing in an enriched or a standard environment. Nerve growth factor-induced gene A and glucocorticoid receptor messenger RNA expression were determined by in situ hybridization two to 30 days after middle cerebral artery occlusion. Stroke induced a decrease in nerve growth factor-induced gene A messenger RNA expression in cortical areas outside the ischemic lesion and in the CA1 subregion of the hippocampus two to three days after ischemia. This decrease was more prolonged with environmental enrichment, lasting until 20 days. However, 30 days after focal cerebral ischemia, environmental enrichment increased nerve growth factor-induced gene A expression compared to standard housing. A reduction of hippocampal glucocorticoid receptor (type II) messenger RNA two to 12 days after stroke in standard housed rats was restored by environmental enrichment. These data suggest that improved functional outcome induced by environmental enrichment after middle cerebral artery occlusion is associated with dynamically altered expression of nerve growth factor-induced gene A messenger RNA in brain regions outside the ischemic lesion, and sustained levels of hippocampal glucocorticoid receptor messenger RNA expression.

Possible roles for His 208 in the active-site region of chloroplast carbonic anhydrase from Pisum sativum.

His 208 of chloroplast pea carbonic anhydrase is conserved among the dicotyledonous carbonic anhydrases. This His was replaced by an Ala to test whether a histidine residue at this position, in analogy to His 64 of human carbonic anhydrase II, acts as an internal proton shuttle. Values of the kinetic parameters kcat and kcat/Km for the H208A mutant enzyme are high over the pH range 6-9 and of the same magnitude as those for the wild-type enzyme, indicating that this residue is not crucial for the catalytic event. The pH dependence of kcat/Km, reflecting the Zn-H2O ionization, is, however, simplified to that of a simple titration with pKa = 7.1 +/- 0.1 in the absence of His 208. Interaction with the proton-accepting buffer molecule is impaired in the mutant, and apparent Km values for the buffer have increased up to 20 times. Furthermore, the H208A mutant is more easily inactivated by oxidation than the wild-type enzyme. The results indicate that the pKa for a redox-sensitive Cys residue is decreased by at least one pH unit in the mutant, and the histidyl side chain seems to have a function in stabilizing the reduced and active form of the enzyme. An interaction with the redox-sensitive cysteines at positions 269 and 272 is proposed.

Chronic amitriptyline treatment induces hippocampal NGFI-A, glucocorticoid receptor and mineralocorticoid receptor mRNA expression in rats.

Adult male rats were treated with the antidepressant drug amitriptyline for 21 days and the expression of specific transcription factors was examined. NGFI-A mRNA expression was increased in the hippocampus and in the cerebral cortex. MR mRNA was increased in the hippocampus while GR mRNA was increased in selective hippocampal regions. There was no change in the NGFI-B mRNA expression. Thus, NGFI-A may be a mediator of plasticity-related phenomena induced by antidepressant drugs.

The sulfhydryl groups of Cys 269 and Cys 272 are critical for the oligomeric state of chloroplast carbonic anhydrase from Pisum sativum.

Chloroplast carbonic anhydrase is dependent on a reducing environment to retain its catalytic activity. To investigate the properties of the three accessible cysteine residues of pea carbonic anhydrase, mutants were made in which Ala or Ser substituted for C165, C269, and C272. The mutants at position 165 were found to be spectroscopically similarly to the wild-type. They have a high catalytic activity, and are also sensitive to oxidation. In contrast, both C269 and C272 were found to be critical both for the structure and for the catalytic activity. All mutants with substitutions at either of these two positions had to be co-overexpressed with GroES/EL chaperones to give soluble enzyme in Escherichia coli. The k(cat) values were decreased by 2 and 3 orders of magnitude for the C272A and C269A mutants, respectively, and the Km values were increased approximately 7 times. However, the binding of the inhibitor ethoxyzolamide was only slightly weakened. The near-UV CD spectra were found to be changed in both sign and intensity compared to that of the wild-type, and the far-UV spectra indicate some loss of alpha-helix structure. Moreover, the quaternary structure was changed from the wild-type octameric to tetrameric in these mutants. The results indicate that mutation of either of these cysteines causes minor structural changes around at least one of the two tryptophans of the subunit. Furthermore, the data demonstrate that C269 and C272 are involved in the interaction between subunits and are necessary for a proper structure at the tetramer-tetramer interface.

High-performance liquid chromatography method for determination of flunixin in bovine plasma and pharmacokinetics after single and repeated doses of the drug.

A high-performance liquid chromatography method was developed for determination of flunixin in bovine plasma. The extraction procedure was easily performed and made it possible to detect low concentrations of flunixin with high accuracy. The limit of quantitation was 7 ng/ml (relative standard deviation = 18% n = 10). The analytic method permits processing of 60 samples/d. Flunixin, as well as the internal standard (diclofenac sodium), belong to the group of nonsteroidal anti-inflammatory drugs, which are known to have a high degree of binding to plasma proteins. Therefore, an evaluation of several buffer systems was undertaken to optimize analytic conditions. Cattle were given 2.2 mg of flunixin melgumine/kg of body weight. In experiment 1, single injections were administered IV to 1 cow and IV and IM to 1 heifer (7 days apart), and pharmacokinetic variables were calculated. The IV data were best described by a two-compartment model. The half-life after single IV or IM administration was around 4.0 hours. In experiment 2, the decreasing flunixin concentration was determined after the last of either 4 IM injections daily (n = 3 cows) or 2 IM injections daily (n = 3 cows) administered during a 14-day postpartum period. The half-life, determined between 48 and 96 hours after the last dose, was approximately 26 hours in both groups, and flunixin could be detected in plasma up to 8 days, on average. The protein binding of flunixin was studied, using the method of equilibrium dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Solvent hydrogen isotope effects and anion inhibition of CO2 hydration catalysed by carbonic anhydrase from Pisum sativum.

Chloroplast carbonic anhydrase from Pisum sativum has been studied to elucidate the catalytic mechanism and to test if the mechanism proposed for human carbonic anhydrase II is also valid for pea carbonic anhydrase. The catalytic activity was found to depend on the chemical nature of the buffer. Barbital buffer gives the highest turnover number at infinite buffer concentration and the lowest Km value with respect to the buffer, while the kinetic parameters obtained in the imidazole-type buffer, 1-methylimidazole, do not differ from those obtained using the biological-type buffer Mops. The anion inhibition of CO2 hydration was investigated using SCN- at pH 6-9. The binding of the anion was found to be pH dependent with the strongest interaction at low pH. We obtained an uncompetitive inhibition pattern at high pH and noncompetitive inhibition patterns at pH 7 and low pH. The catalytic mechanism was further tested by measurements of the solvent hydrogen isotope effects on the kinetic parameters for CO2 hydration. The observed effects were comparatively small with a kcat value of approximately 2 irrespective of the pH. The effect on kcat/Km and on Km changes when going from high pH to pH 7 and low pH. At high pH, the solvent isotope effect in Km is at least 3, giving a value below 1 for kcat/Km, while at pH 7 and low pH the major effect is found in kcat/Km with values of 2.6 and 2.9. The dependence of the CO2-hydration activity on the buffer concentration is in agreement with a ping-pong mechanism with buffer acting as a second substrate. This is analogous to the behaviour of human carbonic anhydrase II. The inhibition patterns and the observed isotope effects at high pH can also be explained within the framework of the catalytic mechanism for human carbonic anhydrase II, with a rate-determining and buffer-dependent part. The results are consistent with a mechanism involving a proton transfer that contributes to rate limitation. However, the isotope effects found at pH 7 and low pH indicate that some part of the mechanism has changed. Moreover, we cannot decide whether the mechanism for pea carbonic anhydrase involves an internal proton-shuttle group, or if the buffer molecule acts in a direct proton transfer from the zinc-coordinated water.

Kinetic studies of pea carbonic anhydrase.

Chloroplast carbonic anhydrase from Pisum sativum has been isolated. The kinetic properties of the enzyme have been studied and comparisons to the well characterised human carbonic anhydrase II made. Pea carbonic anhydrase was found to be dependent on a reducing agent in order to retain the catalytic activity. Oxidised, inactive, enzyme could be activated by the addition of a SH-agent. However, such activation gave only 60% of the activity of an enzyme kept in a reduced state all the time. The kinetics of CO2 hydration show an increase in kcat as well as in kcat/Km with pH, but the pH profile does not follow a simple titration curve. The pH dependence is more complicated and it seems as if there are several titratable groups affecting the activity. At pH 9 we obtain a turnover number of 4 x 10(5) s-1 and a kcat/Km value of 1.8 x 10(8) M-1 s-1 with reference to the subunit. We also find that the enzyme needs high concentrations of buffer to work at a maximal rate. Apparent Km values with respect to the total buffer concentration are found between 52-185 mM at neutral and high pH. At low pH the situation is complex with deviations from Michaelis-Menten kinetics. Chloroplast carbonic anhydrase from higher plants have been reported to have primary structures that are completely different from the enzyme from animals. In addition, we find the circular dichroic spectrum of pea carbonic anhydrase to be well distinguished from that of human carbonic anhydrase II. Despite those structural differences the kinetic parameters indicate that pea carbonic anhydrase is equally efficient as human carbonic anhydrase II in catalysing the hydration of CO2. However, the mechanism for proton transfer from the active site to the surrounding medium seems to differ between the two enzymes.

Determination of theophylline in plasma using different capillary electrophoretic systems.

Theophylline was determined in human plasma by capillary electrophoresis (CE). The drug was used as a model substance to study a simple sample pretreatment often used in HPLC: to 200 microliters of plasma were added 400 microliters of acetonitrile to precipitate the plasma proteins and the supernatant was injected into the capillary after centrifugation. Three capillary electrophoretic systems were compared with respect to migration time and electrophoretic migration reproducibility. UV detection at 280 nm was applied. The separation was preferably made in an uncoated fused-silica capillary (57 cm x 75 microns I.D.) with 10 mM phosphate-borate buffer (pH 9.0) as the electrophoretic buffer. A linear calibration graph was obtained in the concentration range studied, 1.8-36 micrograms/ml (10-200 microM). The method permits the determination of theophylline in plasma at therapeutic concentrations of 4.5-20 micrograms/ml (25-110 microM) with acceptable precision.

Processing of the chloroplast transit peptide of pea carbonic anhydrase in chloroplasts and in Escherichia coli. Identification of two cleavage sites.

The chloroplast transit peptide (cTP) of pea carbonic anhydrase was shown to be processed at two different sites, giving protein subunits of two sizes. The cleavage sites were identified and found to be localized immediately before and after a highly charged part, containing 8 acidic and 6 basic residues, of the cTP. Properties of pea carbonic anhydrase produced in Escherichia coli show that folding, oligomerization and catalytic activity do not depend on the presence of the acidic part or the rest of the cTP. The pattern of processing of the cTP in E. coli indicates that cleavage at site I is specific for a chloroplastic stromal peptidase and that cleavage at site I prevents processing at site II.