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Various approaches have been proposed to denote treatment outcome, such as the effect size of the pre-to-posttest change, percentage improvement, statistically reliable change, and clinical significant change. The aim of the study is to compare these approaches and evaluate their aptitude to differentiate among child and adolescent mental healthcare providers regarding their treatment outcome. Comparing outcomes according to continuous and categorical outcome indicators using real-life data of seven mental healthcare providers, three using the Child Behavior Checklist and four using the Strengths and Difficulties Questionnaire as primary outcome measure. Within each dataset consistent differences were found between providers and the various methods led to comparable rankings of providers. Statistical considerations designate continuous outcomes as the optimal choice. Change scores have more statistical power and allow for a ranking of providers at first glance. Expressing providers' performance in proportions of recovered, changed, unchanged, or deteriorated patients has supplementary value, as it denotes outcome in a manner more easily interpreted and appreciated by clinicians, managerial staff, and, last but not least, by patients or their parents.

Readily accessible and versatile phosphonite building blocks with improved stability against hydrolysis were used for the efficient metal-free functionalization of peptides and proteins in aqueous buffers at low micromolar concentrations. The application of this protocol to the immobilization of a Rasa1-SH2 domain revealed high binding affinity to the human T-cell protein ADAP and supports the applicability of triazole phosphonites for protein modifications without harming their function., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA-synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Context: Two new low-molecular-weight LH agonists (Org 43553 and Org 43902) were shown to induce ovulation in preclinical experiments., Objective: Our objective was to assess the safety, pharmacokinetics, and pharmacodynamics of Org 43553 and Org 43902 when administered to healthy females., Design and Setting: Org 43553 and 43902 studies were randomized, placebo-controlled, single-rising-dose first-in-human trials, which included 159 healthy female volunteers. Part 1 of the studies assessed the safety and pharmacokinetics. Part 2 evaluated the pharmacodynamics effect of a single oral dose of Org 43553 (25-900 mg) or Org 43902 (30-300 mg) to induce ovulation after the development of a large preovulatory follicle, whereas the endogenous LH surge was suppressed due to GnRH antagonist treatment while follicular development was supported with recombinant FSH., Results: Org 43553 and 43902 were safe and well tolerated. Both compounds showed a fast absorption after oral intake, with peak concentrations reached within 0.5 to 1 hour. The elimination half-life of Org 43553 was 30 to 47 hours and that of Org 43902 was 17 to 22 hours. Ovulation induction confirmed by midluteal progesterone rise ≥15 nmol/L was proven in both studies, also when excluding subjects with an endogenous LH rise. The minimal effective dose for ovulation induction was 300 mg in both studies and resulted in an ovulation rate of 83% and 82%, respectively., Conclusions: These first proof-of-concept studies both demonstrated that a single oral intake of an low-molecular-weight LH agonist induces ovulation of the preovulatory follicle in pituitary-suppressed female volunteers of reproductive age.

Chemoselective modification of biomolecules: The reaction between 2-cyanoethyl phosphoramidites and azides is economical and can be performed in different solvents, including aqueous buffers. The course of the reaction with azido-modified amino acids, peptides or proteins and different label molecules was followed by (31)P NMR spectroscopy., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Small fiber neuropathy (SFN) is a disorder typically dominated by neuropathic pain and autonomic dysfunction, in which the thinly myelinated Aδ-fibers and unmyelinated C-fibers are selectively injured. The diagnosis SFN is based on a reduced intraepidermal nerve fiber density and/or abnormal thermal thresholds in quantitative sensory testing. The etiologies of SFN are diverse, although no apparent cause is frequently seen. Recently, SCN9A-gene variants (single amino acid substitutions) have been found in ∼30% of a cohort of idiopathic SFN patients, producing gain-of-function changes in sodium channel Na(V)1.7, which is preferentially expressed in small diameter peripheral axons. Functional testing showed that these variants altered fast inactivation, slow inactivation or resurgent current and rendered dorsal root ganglion neurons hyperexcitable. In this review, we discuss the role of Na(V)1.7 in pain and highlight the molecular genetics and pathophysiology of SCN9A-gene variants in SFN. With increasing knowledge regarding the underlying pathophysiology in SFN, the development of specific treatment in these patients seems a logical target for future studies., (© 2012 John Wiley & Sons A/S.)

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 µg Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.

Objectives: Although small fiber neuropathy (SFN) often occurs without apparent cause, the molecular etiology of idiopathic SFN (I-SFN) has remained enigmatic. Sodium channel Na(v)1.7 is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons and their small-diameter peripheral axons. We recently reported the presence of Na(v)1.7 variants that produce gain-of-function changes in channel properties in 28% of patients with painful I-SFN and demonstrated impaired slow-inactivation in one of these mutations after expression within HEK293 cells. Here we show that the I739V Na(v)1.7 variant in a patient with biopsy-confirmed I-SFN impairs slow-inactivation within DRG neurons and increases their excitability., Methods: A patient with SFN symptoms including pain, and no identifiable underlying cause, was evaluated by skin biopsy, quantitative sensory testing, nerve conduction studies, screening of genomic DNA for variants in SCN9A, and functional analysis., Results: Voltage-clamp analysis following expression within DRG neurons revealed that the Na(v)1.7/I739V substitution impairs slow-inactivation, depolarizing the midpoint (V(1/2)) by 5.6 mV, and increasing the noninactivating component at 10 mV from 16.5% to 22.2%. Expression of I739V channels within DRG neurons rendered these cells hyperexcitable, reducing current threshold and increasing the frequency of firing evoked by graded suprathreshold stimuli., Conclusions: These observations provide support, from a patient with biopsy-confirmed SFN, for the suggestion that functional variants of Na(v)1.7 that impair slow-inactivation can produce DRG neuron hyperexcitability that contributes to pain in SFN. Na(v)1.7 channelopathy-associated SFN should be considered in the differential diagnosis of cases of SFN in which no other cause is found.

The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized proteins containing a site-specifically incorporated azido amino acid was possible using different alkyne-functionalized Ru(II) bathophenanthroline complexes. We were able to achieve a selective labelling even in complex mixtures of proteins despite the fact that ruthenium dyes normally show a high tendency for unspecific interactions with proteins and are commonly used for total staining of proteins. Since the employed Ru complexes are extremely robust, photo-stable and highly sensitive, the approach should be applicable to the production of labelled proteins for single molecule spectroscopy and fluorescence-based interaction studies.

Chemoselective reactions are important tools for the modification of peptides and proteins. Thereby the modification is desired to be site specific and bioorthogonal. Here we describe the site-specific modification of azido-proteins via a Staudinger-type phosphite ligation. The reaction was carried out in aqueous system on proteins containing p-azido-phenylalanine in a single position introduced by the amber codon technique. A selective introduction of branched polyethylene scaffolds can be achieved with the application of the methodology reported herein.

Asenapine is a psychopharmacologic agent approved in the United States for the acute treatment of schizophrenia in adults and the acute treatment of manic or mixed episodes associated with bipolar I disorder with or without psychotic features in adults. It is pending approval for the treatment of schizophrenia and manic episodes associated with bipolar I disorder in Europe. Asenapine is administered as a sublingual formulation. To determine whether the pharmacokinetics of asenapine are impacted by placing the tablet buccally ('cheeking') or allowing the tablet to dissolve on the top of the tongue, pharmacokinetics were compared following buccal and supralingual administration versus sublingual administration. In this open-label, randomized, 3-way crossover trial, healthy men (n=36) received single 5 mg doses of asenapine via sublingual, supralingual and buccal routes, at least 1 week apart. With buccal administration, the area under the concentration-over-time curve (AUC(0-infinity)) and peak concentration (C(max)) were, respectively, 24%, and 19% higher than with sublingual administration; these routes were not bioequivalent. With supralingual administration, AUC(0-infinity) and C(max) were 6% and 13% lower than with sublingual administration; bioequivalence was established based on AUC(0-infinity) only; bioequivalence based on C(max) could not be assessed due to 40% within-subject variability. The most common adverse events were oral paresthesia (sublingual, 75.8%; supralingual, 55.9%; buccal, 45.7%) and somnolence (81.8%; 76.5%; 68.6%). Compared with the recommended sublingual route of asenapine administration, exposure was 24% higher with buccal administration and comparable to supralingual administration. However, differences in exposure associated with variable placement in the oral cavity did not compromise safety in healthy subjects., (2010 John Wiley & Sons, Ltd.)

The genetic code is universal, but recombinant protein expression in heterologous systems is often hampered by divergent codon usage. Here, we demonstrate that reprogramming by standardized multi-parameter gene optimization software and de novo gene synthesis is a suitable general strategy to improve heterologous protein expression. This study compares expression levels of 94 full-length human wt and sequence-optimized genes coding for pharmaceutically important proteins such as kinases and membrane proteins in E. coli. Fluorescence-based quantification revealed increased protein yields for 70% of in vivo expressed optimized genes compared to the wt DNA sequences and also resulted in increased amounts of protein that can be purified. The improvement in transgene expression correlated with higher mRNA levels in our analyzed examples. In all cases tested, expression levels using wt genes in tRNA-supplemented bacterial strains were outperformed by optimized genes expressed in non-supplemented host cells.

Women in the reproductive age are more vulnerable to develop affective disorders than men. This difference may attribute to anatomical differences, hormonal influences and environmental factors such as stress. However, the higher prevalence in women normalizes once menopause is established, suggesting that ovarian hormones may play an important role in the development of depression in women. Ovarian hormones such as estrogen can pass the brain-blood barrier and bind to cytoplasmatic estrogen receptor (ER)-alpha and ER-beta in different areas of the limbic system. During stress, estrogen can modulate the behavioral and neurobiological response depending on the concentrations of estrogen. In this review we present evidence for disparate effects of chronic stress on neuroplasticity and brain activity in male and female rats. Furthermore, we will demonstrate that effects of social support on coping with stress can be mimicked by social housing of rats and that this model can be used for identification of underlying neurobiological mechanisms, including behavior, phosphorylation of CREB and ERK1/2, and brain activity changes as measured with fos expression. Using cyclic administration of estrogen in ovariectomized female rats we could specifically address effects of different plasma estrogen levels and antidepressants on stress-induced neuroplasticity and activity changes. In this model we also studied effects of estrogen on recovery after chronic stress. We conclude that the female brain has a different innate strategy to handle stress than the male brain and that female animal models are necessary for studying the underlying mechanisms and options for treatment.

Prenatal morphine treatment and emotional stress both have been shown to increase sensitivity to reward-related behaviors. It has been postulated that this increased sensitivity to rewarding stimuli may be the result of an enhanced release of endogenous opioids. In the present study, in vivo autoradiography was employed to investigate the endogenous opioid release in specific brain areas in rats. Pregnant animals were exposed to morphine or saline from day 8 of gestation till birth. Development of pups was monitored and play behavior was tested on postnatal day 21. Adult rats were exposed to repeated emotional stress or control treatment for five consecutive days and tested in a small open field 5 days later. [(3)H]-Diprenorphine was injected following this test to investigate endogenous opioid release. Prenatal morphine treatment increased play behavior and endogenous opioid release in a number of cortical and subcortical brain areas after being subjected to an open field challenge later in life. Emotional stress exposure increased locomotor activity in the open field irrespective of the type of prenatal treatment and increased endogenous opioid release in some specific brain areas. It is suggested that the increased release of endogenous opioids in the substantia nigra, the piriform cortex and the septum observed after both types of treatments is related to the increased sensitivity to reward.

Myoclonus-dystonia (M-D) is an autosomal dominantly inherited movement disorder with myoclonic jerks and dystonic contractions most frequently due to a mutation in the epsilon-sarcoglycan (SGCE, DYT11) gene. We describe two unrelated children with M-D (DYT11) who presented with writer's cramp. Due to maternal imprinting the family history appeared initially negative for M-D. In children with writer's cramp screening of the SGCE gene should be considered, even with a negative family history.

It's not easy being green: Real-time visualization of labeled ribosomes and de novo synthesized green fluorescent protein molecules using single-molecule-sensitive fluorescence microscopy demonstrates that the mutant GFPem is produced with a characteristic time of five minutes. Fluorescence of the fastest GFP molecules appears within one minute (see picture).

Objectives: In several Western European countries guidelines regarding urinary incontinence (UI) management in general practice have been drawn up. The aim of this study was to evaluate guideline adherence with feedback in general practice in order to improve UI management., Methods: First, a retrospective audit of female patient records was performed with Egton Medical Information System compared with the NICE Guideline regarding UI. Between 1 January 2000 and 31 December 2006 patients, 18-65 years of age, with newly diagnosed UI (ICPC U04) were included in two general practices in Nottingham, UK. Second, results were presented to the general practitioners (GPs) in a feedback session., Results: Sixty-five patients were identified to have newly reported UI. Gynaecological examination was performed in 51% of cases. Additionally, in 40% of patients a urine sample was obtained for dipstick. Follow-up was performed in 34% of the 65 cases. During a feedback session GPs became aware they do not adhere to these guideline items consistently. Patients often report UI as final after several other problems during one single consultation., Conclusion: Most women with UI were not managed according to the NICE Guideline. Audit feedback created awareness of current UI management in general practice and can therefore be used in improving UI management.

Background and Purpose: Recovery of oculomotor (cranial nerve [CN] III) palsy after surgery of posterior communicating artery (PcomA) aneurysms has been well documented, but recovery after coiling is poorly understood. In this study, we report the recovery after coiling of PcomA aneurysm-induced CN III palsy in 21 patients at follow-up of 1 to 7 years., Materials and Methods: Of 135 patients with a PcomA aneurysm treated with coils between January 1997 and December 2003, there were 21 patients with initial CN III dysfunction who were selected and reevaluated. There were 2 men and 19 women with a mean age of 54.9 years. In 17 patients, CN III palsy was associated with subarachnoid hemorrhage (SAH). Timing of treatment after onset of symptoms was 1 to 3 days in 5 patients, 4 to 14 days in 13, and more than 14 days in 3. Mean size of the aneurysm was 9 mm. Initial CN III palsy was complete in 15 patients and partial in 6. Mean follow-up after coiling was 3.7 years (range, 1-7 years)., Results: Of 15 patients with initial complete CN III palsy, recovery was complete in 3 and partial in 10. In 2 patients, complete CN III palsy was unchanged. Of 6 patients with initial partial CN III palsy, recovery was complete in 5 and partial in 1. Initial partial CN III palsy was the only predictor of complete recovery at follow-up., Conclusion: PcomA aneurysm-induced CN III palsy improves or cures after coiling in most patients. Complete recovery is more likely with initial partial dysfunction of the nerve.

Despite almost a decade of research since the identification of uncoupling protein-3 (UCP3), the molecular mechanisms and physiological functions of this mitochondrial anion carrier protein are not well understood. Because of its highly selective expression in skeletal muscle and the existence of mitochondrial proton leak in this tissue, early reports proposed that UCP3 caused a basal proton leak and increased thermogenesis. However, gene expression data and results from knockout and overexpression studies indicated that UCP3 does not cause basal proton leak or physiological thermogenesis. UCP3 expression is associated with increases in circulating fatty acids and in fatty acid oxidation (FAO) in muscle. Fatty acids are also well recognized as activators of the prototypic UCP1 in brown adipose tissue. This has led to hypotheses implicating UCP3 in mitochondrial fatty acid translocation. The corresponding hypothesized physiological roles include facilitated FAO and protection from the lipotoxic effects of fatty acids. Recent in vitro studies of physiological increases in UCP3 in muscle cells demonstrate increased FAO, and decreased reactive oxygen species (ROS) production. Detailed mechanistic studies indicate that ROS or lipid by-products of ROS can activate a UCP3-mediated proton leak, which in turn acts in a negative feedback loop to mitigate ROS production. Altogether, UCP3 appears to play roles in muscle FAO and mitigated ROS production. Future studies will need to elucidate the molecular mechanisms underlying increased FAO, as well as the physiological relevance of ROS-activated proton leak.

Chronic stress induces neurobiological alterations which have consequences for subsequent stress handling. In the current experiment, ovariectomized rats were subjected daily to a stressor for 21 days. Thereafter, the rats were treated for 21 days with 17beta-estradiol benzoate (10 microg/250 g, once every 4 days) or mirtazapine (10 mg/kg, daily). In this way, we were able to evaluate the ability of these compounds to reverse chronic stress-induced changes in the activity of the limbic system. After 21 days of recovery and treatment, the rats were re-exposed to the adverse environment of the initial stressor and perfused 2 h later. Ovariectomized rats displayed increased numbers of c-Fos-positive nuclei, after re-exposure to the stressor, in the paraventricular nucleus of the hypothalamus, dentate gyrus, medial prefrontal cortex and central and medial amygdala. Cyclic estradiol treatment attenuated the sensitization of the paraventricular nucleus and central amygdala. Mirtazapine increased the number of c-Fos-positive nuclei in the central amygdala and dentate gyrus. Long-term transcriptional changes induced by chronic stress were determined with DeltaFosB immunoreactivity. The medial prefrontal cortex showed an increased number of DeltaFosB-positive nuclei after chronic stress and this was not affected by estradiol or mirtazapine administration during recovery. In conclusion, cyclic estradiol administration reversed chronic stress-induced sensitization in the limbic system, the paraventricular nucleus and central amygdala of female rats, output regions of the limbic system involved in fear responses. Mirtazapine did not achieve this reversal of stress-induced aberrations in the limbic system after 21 days of treatment.

Estradiol modulates stress reactions in female rats. Several studies showed anxiolytic effects of estradiol in behavioral tests, but the underlying mechanisms are still unclear. The aim of the current study was to explore how estradiol-treated rats respond to acute and chronic stress compared to ovariectomized rats. Ovariectomized rats received vehicle or 17beta-estradiol injections (10 microg/250 g) once every 4 days, which induced alternating high and low plasma 17beta-estradiol levels. Stress was presented by daily exposure to an adverse environment in which the animals received five footshocks for either 3 or 22 days. Under control conditions no differences were observed, but as soon as stress was applied, reactions of ovariectomized and estradiol-treated rats diverged. Both acute and chronic stress increased the c-Fos protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Cyclic estradiol treatment reduced this stress-induced activation of the PVN, an effect that seems to be dependent on the plasma estradiol levels. No differences in stress-induced corticosterone responses were revealed between the treatment groups. An increase in the number of ERbeta-expressing cells in the PVN of ovariectomized and estradiol-treated rats during chronic stress implied increased ERbeta-mediated mechanisms during these conditions. The dampening effect of estradiol on the excessive stress-induced activity in the PVN may be beneficial for the animal in its response to chronic recurrent stress by reducing the output of the PVN.

Chronic injections of cocaine (20 mg/kg daily for 10 days) increase activity and decrease anxiety in male C57Bl/6j mice in comparison with animals chronically injected with normal saline. U-50,488H (kappa-opioid receptor agonist; 2.5 mg/kg) produced an anxiolytic effect in animals preinjected with normal saline and had no effect in animals chronically injected with cocaine. Presumably, chronic activation of dopaminergic systems caused by cocaine injections is paralleled by desensitization of kappa-opioid receptor system.

Unlabelled: Social support has a positive influence on the course of a depression and social housing of rats could provide an animal model for studying the neurobiological mechanisms of social support. Male and female rats were subjected to chronic footshock stress for 3 weeks and pair-housing of rats was used to mimic social support. Rats were isolated or housed with a partner of the opposite sex. A plastic tube was placed in each cage and subsequently used as a 'safe' area in an open field test. Time spent in the tube was used as a measurement of anxiety levels. Chronic stress increased adrenal weights in all groups, except for isolated females who showed adrenal hypertrophy in control conditions. In isolated males, chronic stress resulted in an increase in the time the animals spent in the tube. While stress did not affect this parameter in socially housed males, males with a stressed partner showed a similar response as isolated stressed males. Even though adrenal weights showed that isolated females were more affected by stress, after chronic stress exposure, they spent less time in the tube than socially housed females. Socially housed stressed females spent less time in the 'safe' tube compared to control counterparts, indicating that stress has a gender-specific behavioral effect., In Conclusion: pair-housing had a stress-reducing effect on behavior in males. Isolation of females was stressful by itself. Pair housing of females was not able to prevent stress-induced behavioral changes completely, but appeared to reduce the effects of chronic stress.

There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement.

Recent studies have demonstrated that the postnatal development of connections between the basolateral amygdala (BLA) and the medial prefrontal cortex (mPFC) mature around postnatal days 13-15 (pd13-15), whereas these between the BLA and other structures such as the nucleus accumbens and the mediodorsal thalamus are completed by pd7. Accordingly, it is hypothesized that mPFC cytoarchitecture and hence its function may be specifically affected by neonatal (i.e. on pd7) but not later induced (i.e. on pd21) damage to the BLA. To test this hypothesis, rats received excitotoxic lesions to the BLA on either pd7 or pd21 and were subjected to two tests putatively sensitive to mPFC dysfunction, namely food hoarding and spontaneous alternation. In addition, rats were tested for spatial learning and memory, to determine any possible effects on hippocampal function. Consistent with the documented effects of mPFC lesions, pd7 damage to the BLA impaired spontaneous alternation and food hoarding performance, an effect that was not found in rats with BLA lesions induced on pd21. Spatial learning and memory, however, were not affected by the (neonatal) lesion procedure. Together, these results indicate that neonatal BLA damage affects species-specific sequential behavior and flexibility, which may be attributed to abnormal functioning of the mPFC.

Monoamine reuptake inhibitors still reign in the treatment of major depression, but possibly not for long. While medicinal chemists have been able to reduce the side effects of these drugs, their delayed onset of action and considerable non-response rate remain problematic. Of late, serious questions have been raised regarding the efficacy of monoamine reuptake inhibitors. The present review presents an inventory of what is (and until recently was) in the antidepressant pipeline of pharmaceutical companies. Novel antidepressant compounds can be categorised into four groups depending on their target(s): (i) monoamine receptors; (ii) non-monoamine receptors; (iii) neuropeptide receptors; and (iv) hormone receptors. Other possible targets include components of post-receptor intracellular processes and elements of the immune system; to date, however, compounds specifically aimed at these targets have not been the subject of clinical trials. Development of several compounds targeted at monoamine receptors has recently been discontinued. At least five neurokinin-1 (NK(1)) receptor antagonists were until recently in phase II of clinical testing. However, the apparent interest in the NK(1) receptor should not be interpreted as representing a departure from the monoamine hypothesis since neurokinins also modulate monoaminergic systems. In the authors' view, development of future antidepressants will continue to rely on the serendipity-based monoamine hypothesis. However, an alternative approach, based on the hypothesis that chronic stress precipitates depressive symptoms, might be more productive. Unfortunately, clinical results using drugs targeted at components of the HPA axis have not been very encouraging to date. In the short run, the authors believe that augmentation strategies offer the best hope for improving the efficacy of antidepressant treatment. Several approaches to improve the efficacy of SSRIs are conceivable, such as concurrent blockade of monoamine autoreceptors and the addition of antipsychotics, neuromodulators or hormones (HPA axis and gender related). In the long-term, however, construction of a scientifically verified conceptual framework will be needed before more effective antidepressants can be developed. It can be argued that it is not depression itself that should be treated, but rather that its duration should be reduced by pharmacological means. Animal models that take this concept into consideration and identify mechanisms for acceleration of recovery from the effects of stress need to be developed.

Neuroimaging studies in patients suffering from affective disorders have shown decreased volume and reduced regional cerebral blood flow in multiple areas of the prefrontal cortex, including the medial prefrontal cortex and the orbitofrontal cortex. This aberrant brain activity is among other things attributed to chronic stress. Affective disorders occur more often in women than in men. In the current experiment, female mPFC-lesioned and non-lesioned rats were subjected to 3 weeks of chronic unpredictable stress in order to determine the role of the mPFC in dealing with chronic stress, and the consequences of mPFC damage for coping with consecutive stressful events. mPFC damage in female rats intensified the stress-induced activation of the dorsomedial nucleus of the hypothalamus and the paraventricular nucleus of the hypothalamus as measured with Fos expression changes and markedly increased plasma catecholamine levels after 3 weeks of unpredictable stress. Additionally, an mPFC lesion significantly reduced the time of appearance of stress-induced behavioral changes in the open field. Altogether, mPFC dysfunction affects the way female rats react to chronic stress, it not only increased the activation of brain regions involved in neuroendocrine and autonomic responses to stress but it also significantly reduced the time of onset of behavioral changes.

Opioid receptors in the ventral tegmental area, predominantly the mu-opioid receptors, have been suggested to modulate reinforcement sensitivity for both opioid and non-opioid drugs of abuse. The present study was conducted to study signal transduction proteins, which may mediate the functioning of mu-opioid receptors in the neurons of the ventral tegmental area. Therefore, brain slices of the ventral tegmental area were exposed in vitro to the specific mu-opioid agonist fentanyl and immunohistochemically stained for four different activated proteins using phospho-specific antibodies. Fentanyl dose-dependently activated extracellular signal-regulated protein in brain slices of the ventral tegmental area. This activation was reversible with naloxone. Furthermore, naloxone itself also activated extracellular signal-regulated protein kinase. Under the present conditions fentanyl did not affect extracellular signal-regulated protein kinase 1 and 2, Stat and cyclic AMP-response element-binding protein activity. The direct activation of extracellular signal-regulated protein kinase in ventral tegmental area slices by the mu-opioid agonist fentanyl may suggest a role of extracellular signal-regulated protein kinase in reward processes., (Copyright 2003 IBRO)

Western diets consist to a large part of n-6 polyunsaturated fatty acids (PUFAs). These n-6 PUFAs and their conversion products favor immune and inflammatory reactions and compromise vasoregulation, which can contribute to the development of dementia. Recent epidemiological studies associated dementia, particularly the type accompanied by a vascular component, with high, saturated dietary fat intake. Conversely, high fish consumption (a source of long chain n-3 PUFAs) was related to a reduced risk for cognitive decline. Therefore we studied the effects of long chain n-3 PUFAs in rats with bilateral occlusion of the common carotid arteries (2VO), which mimics cerebral hypoperfusion, a risk factor for dementia. Male Wistar rats received experimental diets with a decreased (n-6)/(n-3) ratio from weaning on. At the age of 3 months, the animals underwent 2VO surgery. The rats were tested in the elevated plus maze, an active avoidance paradigm and the Morris water maze (at different survival times). Following behavioral testing, the animals were sacrificed at the age of 7 months. The frontoparietal cortex was analyzed for capillary ultrastructure with electron microscopy. No effects of cerebral hypoperfusion or diet were found on elevated plus maze and active avoidance, while spatial memory in the Morris maze was compromised due to cerebral hypoperfusion under placebo dietary conditions. n-3 PUFA supplementation in combination with extra additives improved the performance of the 2VO animals. The number of endothelial mitochondria, as well as the ratio of microvessels with degenerative pericytes appeared to be lower due to long chain n-3 PUFAs. These results may indicate an improved condition of the blood-brain barrier.

Most Helicobacter pylori strains are susceptible to amoxicillin, an important component of combination therapies for H. pylori eradication. The isolation and initial characterization of the first reported stable amoxicillin-resistant clinical H. pylori isolate (the Hardenberg strain) have been published previously, but the underlying resistance mechanism was not described. Here we present evidence that the beta-lactam resistance of the Hardenberg strain results from a single amino acid substitution in HP0597, a penicillin-binding protein 1A (PBP1A) homolog of Escherichia coli. Replacement of the wild-type HP0597 (pbp1A) gene of the amoxicillin-sensitive (Amx(s)) H. pylori strain 1061 by the Hardenberg pbp1A gene resulted in a 100-fold increase in the MIC of amoxicillin. Sequence analysis of pbp1A of the Hardenberg strain, the Amx(s) H. pylori strain 1061, and four amoxicillin-resistant (Amx(r)) 1061 transformants revealed a few amino acid substitutions, of which only a single Ser(414)-->Arg substitution was involved in amoxicillin resistance. Although we cannot exclude that mutations in other genes are required for high-level amoxicillin resistance of the Hardenberg strain, this amino acid substitution in PBP1A resulted in an increased MIC of amoxicillin that was almost identical to that for the original Hardenberg strain.

Background: Both N-nitroso compounds and colonization with Helicobacter pylori represent known risk-factors for the development of gastric cancer. Endogenous formation of N-nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N-nitroso compounds. Based on experiments with a noncarcinogenic N-nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N-nitrosamines., Materials and Methods: Bacteria were grown in the presence of 0-1000 microM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3., Results: Incubation of Neisseria cinerea (positive control) with 500 microM morpholine and 500 microM nitrite, resulted in a significant increase in formation of N-nitrosomorpholine, but there was no significant induction of N-nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions., Conclusion: H. pylori does not induce formation of the carcinogenic N-nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficulty nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1). From 31 isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 5'-GAAAGC-3' were isolated. Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single-stranded regions of hairpin structures. Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting. Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1. RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system. Aptamers from both groups inhibited this RF1 activity.

Aims: The safety, pharmacodynamics and pharmacokinetics of levormeloxifene, a selective oestrogen receptor modulator (SERM), were investigated in postmenopausal women following single doses and multiple dosing once daily up to 56 days., Methods: The two randomized, double-blind, placebo controlled studies of six single ascending doses and at four multiple dose levels, respectively, included a total of 104 healthy postmenopausal women. Safety assessments comprised vital signs, ECG, haematology, clinical chemistry and reporting of adverse events. The pharmacodynamic properties were investigated after multiple dosing by assessment of the short-term effects on bone and lipid metabolism and on the hypothalamic-pituitary axis. Blood samples for pharmacokinetic analysis were collected at intervals until 648 h (27 days) after single and multiple dosing., Results: Levormeloxifene was tolerated well after single doses in the range of 2.5--320 mg and multiple once daily dosing in the range of 20--160 mg. Adverse events reported were generally mild or moderate. The most frequent adverse events after multiple dosing were headache, abdominal pain and leukorrhea with the highest frequency reported after the highest daily dose of 160 mg levormeloxifene. Five weeks of treatment with 20--160 mg levormeloxifene and 8 weeks of treatment with 40 or 80 mg levormeloxifene reduced the biochemical marker of bone turnover, the collagen I C-terminal telopeptide (CrossLaps) by 44.4% [95% CI: 11.3, 65.1] and 35.5% [95% CI: 14.0, 51.6], respectively, without any dose-dependent decrease in the studied dose range. The total cholesterol and LDL-cholesterol concentrations were significantly reduced by 19--25% and 28--35%, respectively, when compared with placebo. HDL-cholesterol and triglyceride concentrations were not affected. An oestrogen-like effect on the hypothalamic-pituitary axis was observed with approximately 50% reductions of FSH and LH after 8 weeks of treatment. No clinically significant changes of other safety variables were observed. The pharmacokinetic analysis demonstrated a rapid absorption (mean tmax: 2--3 h), a slow elimination (mean t1/2: 4.8--8.4 days) and dose linearity of Cmax and AUC for doses up to 160 mg. As expected for a drug with slow elimination given frequently, the relative fluctuation around the steady state plasma concentration was small and the drug accumulation considerable (RA: 3--5)., Conclusions: Short-term administration of levormeloxifene in postmenopausal women was well-tolerated at doses that elicited a favourable pharmacodynamic response suggesting oestrogen-like bone preserving and antiatherogenic effects. Little variation of peak-trough plasma concentrations was observed during daily administration due to a plasma half-life of approximately 1 week.

Objective: To compare molecular typing by amplified fragment-length polymorphism (AFLP) analysis with pulsed-field gel electrophoresis (PFGE) with respect to the ability to differentiate between epidemiologically related and unrelated isolates of common nosocomial pathogens recovered during a period of endemicity., Design: Retrospective laboratory analysis., Setting: Tertiary-care institution., Methods: 17 isolates of Acinetobacter baumannii, 22 isolates of Pseudomonas aeruginosa, and 22 vancomycin-resistant Enterococcusfaecium (VRE) were typed by both methods., Results: AFLP generated comparable results to PFGE for A baumannii and P aeruginosa isolates; both methods identified epidemiologically related and unrelated isolates. However, strain typing of VRE isolates produced discordant results between the two methods. PFGE identified 10 different strain types and differentiated between all epidemiologically related and unrelated isolates. In contrast, AFLP generated only five different strain types, three of which contained both epidemiologically related and unrelated isolates., Conclusion: Molecular typing by AFLP is comparable to PFGE for A baumannii and P aeruginosa isolates. For VRE isolates, however, PFGE remains the method of choice.

The last decades have shown a rapid increase in nonresponse rates. For this reason it is important to study nonresponse and think about it critically. In this article we investigated whether nonresponse affected estimates of the levels of adjustment problems in children and the correlations between these outcomes. The nonresponse was caused by parents who refused permission to interview their children at school, parents who did not return a questionnaire, teachers who did not complete the questionnaire, and parents who refused to participate in an in-depth study, with nonresponse rates of 9%, 69%, 25%, and 46% respectively. The sample consisted of 1282 children aged 4 5 years and the dependent measures were peer-rated sociometric status, self-rated wellbeing at school, and teacher-rated behaviour problems. Despite considerable nonresponse in some conditions our results showed hardly any evidence for bias. This suggested that bias cannot simply be inferred from the amount of nonresponse and that standard rules such as "nonresponse rates higher than 50% are not acceptable" lack a scientific basis. Instead, we argue that to assess nonresponse bias the specific conditions and analyses of the study will need to be considered and special measures may be required.

A method for simultaneous phenotyping and genotyping for CYP2D6 and CYP2C19 was tested. Six healthy volunteers were selected (three extensive and three poor metabolisers for CYP2D6). CYP2D6 was probed with dextromethorphan and metoprolol and CYP2C19 was probed with omeprazole. Blood samples were collected and analysed for dextromethorphan, dextrorphan, metoprolol, alpha-hydroxymetoprol, omeprazole and 5-hydroxyomeprazole by HPLC. Genotyping was performed for both CYP2D6 and CYP2C19. Generally, plasma levels could be measured up to 8 h post-dose except for alpha-hydroxymetoprolol in poor metabolizers (PMs) and dextromethorphan in extensive metabolizers (EMs) (35% below quantification limit). The correlation between the metabolic ratio based on timed individual measurements and the metabolic ratio based on the AUC0-12 values was significant at 3 h post-dose for all probes. In conclusion, the following procedure is suggested: administer metoprolol (100 mg) and omeprazole (40 mg); after 3 h, take a blood sample to assess the genotype and the metabolic ratio for CYP2D6 (metoprolol over alpha-hydroxymetoprolol) and CYP2C19 (omeprazole over 5-hydroxyomeprazole) in plasma. With this procedure, all necessary information on the individual CYP2D6 and CYP2C19 metabolising capacity can be obtained in a practical, single-sample approach.

The discovery of endogenous opioids has markedly influenced the research on the biology of addiction and reward brain processes. Evidence has been presented that these brain substances modulate brain stimulation reward, self-administration of different drugs of abuse, sexual behaviour and social behaviour. There appears to be two different domains in which endogenous opioids, present in separate and distinct brain regions, are involved. One is related to the modulation of incentive motivational processes and the other to the performance of certain behaviours. It is concluded that endogenous opioids may play a role in the vulnerability to certain diseases, such as addiction and autism, but also when the disease is present, such as alcoholism.

The consequences of juvenile isolation and morphine treatment on general activity, social activity and endogenous opioid release during a social interaction test were investigated in the adult rat. Rats were either isolated or socially housed during weeks 4 and 5 of age and treated daily during this isolation period subcutaneously with either saline or morphine. Directly after a social interaction test at 10 weeks of age, rats were injected with [3H]-diprenorphine and subsequently prepared for in vivo autoradiography. The autoradiographic technique was used to visualise neuroanatomical changes in opioid receptor occupancy, probably reflecting changes in opioid peptide release, as a result of social activity. Juvenile isolation increased general activity during the social interaction test, an effect which was accompanied by a reduction of opioid receptor occupancy in many brain areas, suggesting an increased opioid peptide release as a consequence of socially-induced general activity. Morphine treatment in isolated rats caused an increase in adult social activity and enhanced opioid peptide release in some cortical regions and the ventral tegmental area as compared to saline treated rats. Both social activity and opioid receptor occupancy were unaffected by morphine treatment in non-isolated rats. The present study underscores the role of opioid systems in adult social behaviors as a consequence of juvenile isolation. The results suggest a relationship between social activity and opioid peptide release during social contact. Increased social activity seems to be accompanied by elevated opioid peptide release in distinct brain areas after morphine treatment during juvenile isolation., (Copyright 1999 Elsevier Science B.V.)

Acid resistance is considered an important virulence factor of the human pathogen Helicobacter pylori. The enzyme urease plays an important role in this acid resistance, but there are indications that other systems are present. We set out to establish the relevance of these urease-independent acid-resistance systems for growth at low pH. Four mutants out of a total of 1000 UV-mutants were urease positive, grew identical to wild-type on pH 7 plates, but did not grow on pH 5 plates. Whereas transformation of a mutant with its own chromosomal DNA did not restore growth at pH 5, transformation with wild-type DNA or DNA of one of the other mutants did restore the growth. From these complementation studies, we conclude that in H. pylori a urease-independent acid-resistance system, probably depending on the expression of more than one gene, is involved in the growth at low pH.

The modulation of the reinforcing effects of cocaine by the kappa-opioid receptor antagonist, nor-binaltorphimine was studied by using the initiation of intravenous self-administration in drug-naive Wistar rats. Treatment with nor-binaltorphimine (3.0 mg/kg s.c.) 48 h before the start of the first of five daily self-administration sessions significantly decreased the intake of cocaine when offered in a threshold unit dose (30 micrograms per infusion), but had no effect on cocaine intake when it was offered in a higher unit dose (60 micrograms per infusion). It is concluded that blockade of the kappa-opioid receptor by nor-binaltorphimine may produce a rightward shift of the unit dose-response relationship of cocaine reward, thus decreasing the sensitivity to cocaine reward. These data suggest an involvement of endogenous kappa-opioid systems in the mechanisms underlying the initiation of cocaine self-administration behaviour.

The fluorescent probes laurdan (6-dodecanoyl-2-dimethylaminonapthalene) and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl] dipalmitoyl-L-alpha-phosphatidylethanolamine (NBD-PE) in addition to Fourier transform infrared spectroscopy (FTIR) were employed to measure the phase behavior and physical properties of hepatocyte plasma membranes isolated from the livers of thermally acclimated (5 and 20 degreesC) rainbow trout (Oncorhynchus mykiss). The primary objective was to determine the extent to which the phase behavior of membrane lipids is conserved at different growth temperatures. Arrhenius plots of laurdan-generalized polarization revealed a single discontinuity believed to reflect either the onset of the gel-fluid phase transition or the formation of gel phase microdomains, and this discontinuity occurred at significantly higher temperatures in membranes of 20 degrees C (13.2 +/- 0.7 degrees C)- than 5 degrees C (7.2 +/- 0.1 degrees C)-acclimated trout. Similarly, acclimation from 5 to 20 degrees C increased both the onset temperature (from 2.0 +/- 0.3 to 7.2 +/- 0.6 degrees C) and the thermal range (from 10.9 +/- 0.5 to 16.0 +/- 1.0) of the gel-fluid transition as assessed by FTIR. The gel-fluid transition midpoint (approximately -2 degrees C) and completion temperatures (-9 degrees C) were unchanged by thermal acclimation. The anisotropy of NBD-PE fluorescence displayed a distinct minimum in membranes of both warm- and cold-acclimated trout (reflecting alterations in lipid packing that in pure lipid membranes ultimately lead to the formation of nonlamellar phases) in the range of 56-58 degrees C; only membranes of 5 degrees C-acclimated trout displayed an additional minimum at significantly lower temperatures (24.5 +/- 1.7 degrees C). Collectively, these data suggest that the regulation of both the temperature at which gel phase lipids begin to form in response to cooling as well as the propensity of membrane lipids to form nonlamellar phases at higher temperatures may be key features of membrane organization subject to adaptive regulation.

A 74-year-old woman suffered from severe gustatory sweating and flushing of the preauricular skin following parotidectomy (Frey's syndrome). She was treated with intracutaneous botulinum A toxin injections in the affected skin area. Minor's test was used to determine the extent of the affected area. Within one week, the symptoms disappeared. After three weeks, Minor's test was repeated and showed minimal residual hyperhidrosis. These small areas were treated again. No side effects were noted. At follow-up one year later, the patient was free of symptoms.

In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of Helicobacter pylori NCTC 11638 which confers to a siderophore synthesis mutant of Escherichia coli (EB53 aroB hemA) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the ribA gene of Bacillus subtilis. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designated ribBA) resulted in the formation of one translational product, which was able to complement both the ribA and the ribB mutation in E. coli. Expression of ribBA was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin in H. pylori cells is iron regulated. E. coli EB53 containing the plasmid with H. pylori ribBA excreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by E. coli EB53 transformed with the H. pylori ribBA gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of ribBA in H. pylori eliminates its ferric-iron-reduction activity, we conclude that ribBA has an important role in ferric-iron reduction and iron acquisition by H. pylori.

The administration of moderate to high doses of corticosteroids is associated with bone loss. This probably results from the uncoupling of bone formation (decreased) and bone resorption (unchanged or increased). We examined the effect of low-dose (10 mg/day) prednisone (LDP) and the possible mitigating effects of calcium and 1.25 (OH)2 vitamin D (calcitriol) on calcium and bone metabolism in eight healthy, young male volunteers. The study consisted of four observation periods: in the first period, LDP was prescribed during 1 week; in the second, third and fourth periods, calcium (500 mg/day), calcitriol (0.5 micrograms b.i.d.) and calcium in combination with calcitriol, respectively, were added to LDP. Bone formation was measured by means of serum osteocalcin, carboxy-terminal propeptide of type 1 procollagen (P1CP) and alkaline phosphatase, bone resorption by means of urinary excretion of calcium, hydroxyproline, (free and total) pyridinoline, (free and total) deoxypyridinoline and serum carboxy-terminal cross-linked telopeptide of type 1 collagen (1CTP). Dietary calcium and sodium intake were maintained at a stable level during the entire study period. Treatment with LDP led to a decrease in osteocalcin, P1CP and alkaline phosphatase (all P < 0.01). Urinary excretion of pyridinolines, hydroxyproline and serum 1CTP did not increase, but remained unchanged or slightly reduced (P < 0.05), depending on the time of measurement and the marker of bone resorption. Parathyroid hormone (PTH) (insignificantly) increased during LDP (+19%) and LDP plus calcium (+14%), but decreased during supplementation with calcitriol (-16%) and calcium/calcitriol (-44%; P < 0.01). Urinary excretion of calcium increased during treatment with LDP and calcitriol (P < 0.05) and calcium/calcitriol (P < 0.05). It is concluded that LDP has a negative effect on bone metabolism, since bone formation decreased while bone resorption remained unchanged or decreased slightly. The increase in PTH during LDP could be prevented by calcitriol combined with calcium supplementation.