A novel fibrinogen γ chain frameshift deletion (c.637delT) in a patient with hypodysfibrinogenemia associated with thrombosis.

UNLABELLED: Inherited fibrinogen (FG) disorders are rare and result in quantitative or/and qualitative FG deficiency. While the majority of patients with clinically relevant FG deficiencies demonstrate a bleeding phenotype, a subset of patients are at increased risk of thrombosis. PATIENTS AND METHODS: We report a 54-years old man presenting with a thrombophilic phenotype characterized by two episodes of unprovoked venous thrombosis and a deep vein thrombosis several weeks after myocardial infarction. Recently, he developed A. carotis communis thrombosis and died. Coagulation tests were done using standard procedures. FG genes were screened using direct sequencing. Effect on fibrin clot structure was analyzed by scanning electron microscopy (SEM) and FG chain polymerization was analysed using SDS-PAGE. RESULTS: While thrombophilia testing was negative, we found a decreased concentration of clottable FG (126-148 mg/dl) compared to FG antigen (182-194 mg/dl of normal). The thrombin time was slightly prolonged, while aPTT and reptilase time were within the normal range. A novel deletion in FGG gene (c.637delT) resulting in a frameshift and the premature termination of the γ chain at amino acid position p.228 was identified. SDS-PAGE showed a time-shift in γ-γ and α-α cross linking. SEM showed no statistically significant differences between the patient´s and a healthy control´s fibrin clot structure. CONCLUSIONS: In addition to the reduction of FG concentration expected by the nature of the mutation also a functional defect (hypodysfibrinogenemia) was found. Moreover this mutation seems to increase the risk of thrombosis warranting long term anticoagulation possibly in a combination with antiplatelet drugs.

Pharmacodynamic effects of aripiprazole and ziprasidone with respect to p-glycoprotein substrate properties.

INTRODUCTION: Aripiprazole, an atypical antipsychotic drug with mixed antagonism and agonism on dopamine D2 and serotonin receptors, is a substrate of the efflux transporter P-glycoprotein (P-gp). Here we tested the pharmacodynamic consequences of these properties in a P-gp deficient mouse model by studying the effects of aripiprazole and of ziprasidone on motor coordination. METHODS: The motor behaviour of wild-type (WT) and P-gp deficient [abcb1ab(-/-)] mice was investigated on a RotaRod. Mice received acute injections of either aripirazole or ziprasidone. For comparison, the dopamine receptor antagonist haloperidol and serotonin receptor ligands buspirone and ketanserin were also applied. RESULTS: Pharmacokinetic analyses revealed P-gp activity for aripiprazole and ziprasidone. This was indicated by 3.1- and 1.9-fold higher ratios of brain to plasma concentrations of drugs in knock-out to WT animals. Acute doses of ariprazole or ziprasidone impaired motor behaviour on the RotaRod. Effects were similar after injection of haloperidol, whereas the serotonin receptor ligands buspirone and ketanserin enhanced RotaRod performance. Genotype dependent differences of motor performance were found for aripiprazole but not for ziprasidone. DISCUSSION: Evidence was given that P-gp substrate properties have pharmacodynamic consequences for aripiprazole but not for ziprasidone and thus affect dopamine receptor related motor behaviour.

Behavioural consequences of p-glycoprotein deficiency in mice, with special focus on stress-related mechanisms.

P-glycoprotein (P-gp), an efflux transporter localised in the blood-brain barrier, limits the access of multiple xenobiotics to the central nervous system. Whether it is also implemented in the transport of the endogenous glucocorticoid corticosterone is a matter of debate. The P-gp knockout mouse model [abcb1a/b (-/-)] has been shown to differ in the functioning of the hypothalamic-pituitary adrenal (HPA) axis. In the present study, we investigated the behaviour of abcb1a/b (-/-) and wild-type mice with respect to stress-related tests and the effects of corticosterone. Behavioural activities were assessed in the open field (OF) test for 4 days, and in the forced swimming test (FST) and tail suspension test (TST) under naïve and stressed conditions. The FST was also conducted after exogenous corticosterone injection (0.25 and 2.5 mg/kg). Moreover, the elevated plus maze test and the RotaRod test (RotaRod Advanced; TSE Systems, Bad Homburg, Germany) were assessed. Brain corticosterone levels were determined by an immunoassay and expression of glucocorticoid receptors by western blot analysis. Abcb1a/1b (-/-) mice showed significantly decreased brain corticosterone levels and elevated glucocorticoid receptor expression. Behavioural analysis revealed a significantly decreased activity in the OF test on the first 2 days in abcb1a/1b (-/-) mice compared to wild-type mice, although the differences disappeared under habituation. Immobility time in the FST was significantly decreased in abcb1a/1b (-/-) mice under basal and under stressed conditions, whereas immobility in the TST was significantly elevated in these mice under all conditions. Injection of exogenous corticosterone resulted in significant reductions of immobility in the FST in abcb1a/1b (-/-) mice, whereas wild-type mice did not respond to the same doses. There were no differences in the elevated plus maze test and RotaRod test. The results obtained in the present study demonstrate that a P-gp deficiency has an impact on the stress-related behaviour, possibly as a result of differences in HPA axis-feedback regulation.

Spatial learning and expression patterns of PP1 mRNA in mouse hippocampus.

BACKGROUND: Synaptic plasticity is believed to be the major cellular basis for learning and memory. Protein phosphorylation is a key process involved in changes in the efficacy of neurotransmission. In long-term changes synaptic plasticity is followed by structural plasticity and protein de novo synthesis. Such mechanisms are believed to build the basis of hippocampal learning and memory investigated in the Morris water maze (MWM) task. To examine the role of dephosphorylation during that model for spatial learning, we analyzed protein phosphatase 1 (PP1) expression in the hippocampus of mice at various stages of the task and in two groups with different learning abilities. METHODS: Mice were trained for 4 days with four trials each day in the MWM. For gene expression hippocampi were prepared 1, 6 and 24 h after the last trial of each day. PP1 and brain-derived neurotrophic factor (BDNF) mRNA levels were determined by quantitative real-time PCR. RESULTS: The task requirements themselves affected expression levels of both PP1 and BDNF. In contrast to BDNF, PP1 was differentially expressed during learning. Poorly and well performing mice differed significantly. When performance was poor the expression level of PP1 was higher. CONCLUSION: Present results add further in vivo evidence that not only phosphorylation but also dephosphorylation is a major mechanism involved in learning and memory. Therefore, inhibition of hippocampal phosphatase activity might improve learning and memory.

Detection of behavioral alterations and learning deficits in mice lacking synaptophysin.

The integral membrane protein synaptophysin is one of the most abundant polypeptide components of synaptic vesicles. It is not essential for neurotransmission despite its abundance but is believed to modulate the efficiency of the synaptic vesicle cycle. Detailed behavioral analyses were therefore performed on synaptophysin knockout mice to test whether synaptophysin affects higher brain functions. We find that these animals are more exploratory than their wild type counterparts examining novel objects more closely and intensely in an enriched open field arena. We also detect impairments in learning and memory, most notably reduced object novelty recognition and reduced spatial learning. These deficits are unlikely caused by impaired vision, since all electroretinographic parameters measured were indistinguishable from those in wild type controls although an inverse optomotor reaction was observed. Taken together, our observations demonstrate functional consequences of synaptophysin depletion in a living organism.

Cyclosporine A (CsA) affects the pharmacodynamics and pharmacokinetics of the atypical antipsychotic amisulpride probably via inhibition of P-glycoprotein (P-gp).

The importance of P-glycoprotein (P-gp) in the pharmacokinetics of amisulpride and the effects of a P-gp inhibitor cyclosporine A (CsA) was investigated both, in vitro and in vivo. In vitro and in vivo results indicated amisulpride as a substrate of P-gp. Amisulpride was not metabolized by rat liver microsomes. Open field behavior showed time dependent abolishment in locomotion by amisulpride (50 mg kg(-1)). Co-administration of CsA (50 mg kg(-1)) resulted in a higher and significantly longer antipsychotic effect (24 h after drug administration). Accordingly, the area under concentration-time curve in serum and brain was higher in CsA co-treated rats (13.5 vs. 29.8 micromol h l(-1) for serum and 2.16 vs 2.98 micromol h l(-1) for brain tissue) while renal clearance was not affected. These results pointed to a pharmacokinetic drug interaction between CsA and amisulpride most likely caused by inhibition of P-gp.

Topochemical studies on modified lignin distribution in the xylem of Poplar (Populus spp.) after wounding.

BACKGROUND AND AIMS: Information on the influence of wounding on lignin synthesis and distribution in differentiating xylem tissue is still scarce. The present paper provides information on cell modifications with regard to wall ultrastructure and lignin distribution on cellular and subcellular levels in poplar after wounding. METHODS: Xylem of Populus spp. close to a wound was collected and processed for light microscopy, transmission electron microscopy and cellular UV microspectrophotometry. Cell wall modification with respect to lignin distribution was examined at different stages of wound tissue development. Scanning UV microspectrophotometry and point measurements were used to determine the lignin distribution. KEY RESULTS: Xylem fibres within a transition zone between differentiated xylem laid down prior to wounding and the tissues formed after wounding developed distinctively thickened secondary cell walls. Those modified walls and cell corners showed, on average, a higher lignin content and an inhomogeneous lignin distribution within the individual wall layers. CONCLUSIONS: The work presented shows that wounding of the xylem may induce a modified wall architecture and lignin distribution in tissues differentiating at the time of wounding. An increasing lignin content and distinctively thickened walls can contribute to improved resistance as part of the compartmentalization process.

Reduction of the diagnostic window in three cases of human immunodeficiency-1 subtype E primary infection with fourth-generation HIV screening assays.

BACKGROUND AND OBJECTIVES: The influence of genetic variability on the sensitivity of serological diagnosis of human immunodeficiency virus (HIV) infection has, to date, been poorly investigated. The aim of the present study was to assess whether fourth-generation assays for the combined detection of HIV antigen and antibodies to HIV (anti-HIV) permit a reduction of the diagnostic window in comparison to third-generation antibody enzyme immunoassays (EIAs), which so far have shown a poor sensitivity for detection of HIV-1 non-subtype B primary infections. MATERIALS AND METHODS: Three patients with primary HIV-1 subtype E (CRF01-AE) infection were tested with different third- and fourth-generation assays, stand-alone HIV antigen (Ag) EIAs and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, virus lysates from HIV-1 Group M and O and HIV-2, at concentrations of p24 Ag close to the detection limit of licensed HIV Ag EIAs, were investigated with fourth-generation EIAs and HIV Ag EIAs. RESULTS: In the first blood donor, the most sensitive fourth-generation assay detected HIV-1 infection 11 days earlier than five of the eight third-generation antibody assays. Fourth-generation EIAs, with a high sensitivity for HIV antigen, detected HIV-1 subtype E infection simultaneously or 4 days later than HIV-1 RT-PCR on pooled samples. Low concentrations of virus lysates of different HIV-1 subtypes A-H and group O, tested positive with fourth-generation EIAs, with a high sensitivity of the antigen-detection module. CONCLUSIONS: Fourth-generation EIAs, especially those with a high sensitivity for HIV-1 p24 antigen, reduce the diagnostic window for primary HIV-1 subtype E infection in comparison with third-generation antibody-screening assays. These preliminary data from seroconversions and virus lysates indicate that the genetic diversity of HIV-1 does not represent a major challenge for the most sensitive EIAs of this new assay generation.

Developmental stages and fine structure of surface callus formed after debarking of living lime trees (Tilia sp.).

Wounding of trees by debarking during the vegetative period sometimes results in the formation of callus tissue which develops over the entire wound surface or on parts of it. This light and transmission electron microscopy study of living lime trees found that the formation of such a surface callus is subdivided into three stages. During the first stage, numerous cell divisions take place in regions where differentiating xylem remains at the wound surface after debarking. This young callus tissue consists of isodiametric parenchymatous cells. Cambium cells, sometimes also remaining at the wound surface, collapse and do not contribute to callus formation. During the second stage, cells in the callus undergo differentiation by forming a wound periderm with phellem, phellogen and phelloderm. In the third stage, a cambial zone develops between the wound periderm and the xylem tissue laid down prior to wounding. This process is initiated by anticlinal and periclinal divisions of a few callus cells only. Later this process extends tangentially to form a continuous belt of wound cambium. Subsequently, this cambium produces both wound xylem and wound phloem and thus contributes to further thickening.

Anxiolytic-like effects of acute and chronic GABA transporter inhibition in rats.

Acute GABA transporter inhibition can induce anxiolytic-like behaviors. The present analysis addressed whether chronic treatment (23 days via drinking water) with a GABA transporter inhibitor affects rat behavior similar to acute treatment and interferes with additional benzodiazepine-receptor agonistic treatment. Seventy-one rats divided into seven groups were acutely treated with either vehicle, diazepam (2 mg/kg), zolpidem (0.05 mg/kg), tiagabine (19 mg/kg) or chronically with tiagabine with or without acute diazepam or zolpidem. Animals were behaviorally characterized in an elevated plus-maze. None of the treatments induced changes in the activity of the animals. Acute and chronic treatment with tiagabine induced anxiolytic-like effects, similar to acute doses of diazepam. Acute diazepam did not enhance chronic tiagabine effects, whereas acute zolpidem attenuated the anxiolytic-like effects of chronic tiagabine. It is concluded that anxiolytic effects of acute GABA-uptake inhibition by tiagabine persist under chronic treatment and are sensitive to concomitant use of benzodiazepine receptor ligands.

Serum CEA and CA 15-3 as prognostic factors in primary breast cancer.

In the present study, we investigated the association of the serum levels of the tumour markers carcinoembryonic antigen and cancer antigen 15-3 with disease free survival and death from disease in 1046 women with breast cancer without metastases at the time of primary diagnosis in relation to age and the established prognostic factors tumour size, lymph node status, histological grading and hormone receptor status. We found that elevated pre-operative serum marker values were correlated with early relapse (cancer antigen 15-3; P=0.0003) and death from disease (carcinoembryonic antigen, cancer antigen 15-3; P=0.0001 both) in univariate analyses. By comparing pre- and post-operative values we found a decline in values post-surgery. In those patients where marker levels of carcinoembryonic antigen decreased more than 33%, a significantly higher risk for relapse and death from disease (both P=0.0001) in univariate analyses was observed. In multivariate analysis this decrease of carcinoembryonic antigen proved to be an independent prognostic factor. The results for cancer antigen 15-3 were comparable to carcinoembryonic antigen in univariate analyses but showed no significance in multivariate analysis. In this study the post-operative decrease of the serum tumour marker carcinoembryonic antigen was a strong independent prognostic factor for disease free survival and death from disease in breast cancer patients.

The significance of Tenascin-C serum level as tumor marker in squamous cell carcinoma of the head and neck.

BACKGROUND: Tenascin, an extracellular matrix glycoprotein, is transiently present in embryonic tissue, in benign granulation tissue, but also in several highly anaplastic tumors like fibrosarcoma, melanoma and squamous cell carcinoma of the skin. This study was performed to validate elevated Tenascin serum levels as a possible marker for head and neck squamous cell carcinomas (HNSCC). PATIENTS AND METHODS: Tenascin serum levels were evaluated in patients with primary (n = 92) and with recurrent (n = 28) HNSCC. Patients with benign, non inflammatory ear, nose and throat diseases (n = 16) served as the control. The Tenascin serum levels were measured by ELISA (Aventis). RESULTS: Serum Tenascin concentrations of patients with benign ENT diseases ranged between 0.37 and 2.19 micrograms/ml (n = 16, mean +/- SD: 1.23 +/- 0.59 micrograms/ml), of patients with HNSCC (primary diagnosis) between 0.05 and 8.75 micrograms/ml (n = 92, mean +/- SD: 1.81 (1.36 micrograms/ml) and of patients with recurrent HNSCC between 0.53 and 10.0 micrograms/ml (n = 28, mean +/- SD: 2.78 +/- 2.2 micrograms/ml). CONCLUSION: We found a significant elevation of Tenascin serum levels only in patients with higher tumor stages (T4/UICC4) (p < 0.01/p < 0.1) or recurrent disease compared to Tenascin serum levels in healthy controls. Thereby Tenascin serum levels cannot be used clinically as a routine serum marker for the control of head and neck cancer. Further investigations are necessary to evaluate whether the measurement of Tenascin levels as tumor markers could offer additional information to the clinical outcome of patients with HNSCC.

Ragout-jet FTIR spectroscopy of cluster isomerism and cluster dynamics: from carboxylic acid dimers to N2O nanoparticles.

Direct absorption supersonic jet Fourier transform spectroscopy provides a panoramic view of the dynamics of molecular clusters over the entire IR spectral range. The new and generally applicable ragout-jet technique compensates for the sensitivity limits inherent in the incoherent FTIR approach by the use of synchronized giant gas pulses expanding into a large vacuum buffer. A modification based on fragmented interferograms is proposed and demonstrated, by which the spectral resolution can be extended to the limit of the available FTIR spectrometer. The power of the method is illustrated for two classes of compounds. For acetic acid and its isotopomers, the supersonic jet spectra of dimers and oligomers are investigated for the first time, concentrating on the very complex OH/CH stretching domain and on the more regular C=O/C-O stretching range. Issues of cluster isomerism, hydrogen exchange tunneling, anharmonic resonances, intermolecular Franck-Condon sequences, methyl group substitution and cluster coating with argon are explored. For the more weakly interacting nitrous oxide, stretching fundamentals and combination bands of clusters in the 1-3 nm range are studied as a function of composition. Surface vibrations are investigated in detail and modeled quantum mechanically. The semiempirical AM1 approach is found to provide a remarkably accurate description of the cluster structure, energetics and dynamics.

Enantioseparation of chiral thiobarbiturates using cyclodextrin-modified capillary electrophoresis.

The racemates of several chiral thiobarbiturates were separated by using different cyclodextrins in capillary electrophoresis (CE). Six neutral and negatively charged cyclodextrins 1 (CDs) were employed as chiral separators whereof five led to successful separation of enantiomeric thiobarbiturate pairs. The CDs used were the native alpha-CD, beta-CD, gamma-CD, and heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (HDM) as well as heptakis-(2,3-di-O-methyl-6-sulfato)-beta-cyclodextrin (HDMS) and heptakis-(2,3-di-O-acetyl-6-sulfato)-beta-CD (HDAS). Five of the six chiral thiobarbiturates studied could be resolved at a basic pH value of 9.4 and a phosphate buffer concentration of 100 mM in a fused-silica capillary. Structurally related substances showed a similar behavior in separation: 1 and 2 bearing the center of chirality in the side chain at C5 can be best separated using gamma-CD, the N-alkyl-substituted compounds 3 and 4 as well as the N/S-dialkyl-substituted compound 5 could be resolved with HDM. Using the neutral CDs, the migration times were relatively small (< 11 min). 3 and 4 could be also resolved by means of the negatively charged HDMS. In the latter case, the migration time is twice as long as with HDM.

Behavioral analysis indicates benzodiazepine-tolerance mediated by the benzodiazepine binding-site at the GABA(A)-receptor.

1. GABA(A)-receptor induced changes in locomotion and anxiety-like behaviors were studied in rats using an open-field and an elevated plus-maze. Acute and chronic doses of the benzodiazepine diazepam without and in combination with the GABA uptake inhibitor SKF-89976A were investigated. 2. Fifty-six male rats of the strain PVG/OlaHsd (PVG; 180-200 g body wt) were used to assess the influence of the benzodiazepine binding-site to the development of tolerance. Rats were divided into six groups: The first receiving saline (0.9%), the second and third diazepam (10.0 mg/kg) daily for 23 days with or without an acute challenge of 2.0 mg/kg diazepam. The fourth group received diazepam (10.0 mg/kg) daily and acutely SKF-89976A (15.0 mg/kg) plus diazepam and the fifth and sixth group received acute treatment with diazepam (2.0 mg/kg) or SKF-89976A (15.0 mg/kg). 3. Under chronic treatment with diazepam the animals became tolerant to acute doses of diazepam in activity and anxiety-related behaviors. Acute treatment with SKF-89976A increased exploration. Parameters expressing anxiolytic-like behaviors were increased, too, but not all of them significantly. In diazepam tolerant animals SKF-89976A produced anxiolytic-like behaviors 4. We conclude that the BZ- and not the GABA-binding site at the GABA(A)-receptor is involved in the development of BZ-tolerance.

Numerical aspects of spatio-temporal current density reconstruction from EEG-/MEG-data.

The determination of the sources of electric activity inside the brain from electric and magnetic measurements on the surface of the head is known to be an ill-posed problem. In this paper, a new algorithm which takes temporal a priori information modeled by the smooth activation model into account is described and compared with existing algorithms such as Tikhonov-Phillips.

Spatio-temporal current density reconstruction (stCDR) from EEG/MEG-data.

Among the different approaches to the bioelectromagnetic inverse problem, the current-density reconstruction methods (CDR) provide the most general solutions. Since the inverse problem does not have a unique solution, model assumptions have to be taken into account. Multi-channel measurements contain not only spatial, but also temporal information about the sources, so a naturally extension to existing methods leads to spatio-temporal model constraints. Spatio-temporal CDR's (stCDR) have been tested in simplified volume conductor models, assuming different spatial model constraints and a smooth temporal activation model. Comparison to existing spatial model constraints showed a significant improvement of spatial and temporal resolution of the reconstructed sources for the spatio-temporal models especial in noisy data.

Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase.

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE-Tg mice as compared with controls. Also, the high-affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE-Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE-Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus-maze test. Systemic application of a low dose of physostigmine (100 microgram/kg i.p.) normalized all of the enhanced parameters in hAChE-Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE-Tg mice, hippocampal ACh release is up-regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.

The formation and dynamics of proton wires in channel environments.

By virtue of an accurate interaction model, the equilibrium and dynamical properties of an excess proton in aqueous systems are studied, in which the water and excess proton are confined to hydrophobic cylindrical channels. Solvation structures of the excess proton and its mobility along the channel are considered as a function of the channel radius. It is found that when the aqueous proton systems are sufficiently constricted there is a substantial increase in the diffusion of the excess proton charge accompanied by a decrease in the diffusion of water molecules along the channel. Such systems present clear evidence for the possible existence of "proton wires."

Comparison between a nucleic acid sequence-based amplification and branched DNA test for quantifying HIV RNA load in blood plasma.

HIV RNA was quantified in blood plasma from 209 patients and in control specimen comparing the NucliSens HIV-1 QT test (Organon Teknika), which is based on the nucleic acid sequence amplification procedure, and the Quantiplex 3.0 test (Bayer), which uses hybridization signal enhancement by branched DNA (bDNA) probes. A highly significant correlation (P=0.01) was found between the two methods with 88% of the samples showing similar results. In cases of discrepant findings, higher virus load was observed with either test (14xNASBA>bDNA; 12xbNDA>NASBA). Differences could neither be related to clinical features nor to divergent virus subtypes. Standard preparations containing 35000 and 222000 copies were quantified with intra-assay coefficients of variation of <20% using both methods. A preparation of 192 copies was measured with lower precision by both tests, yet was detected more reliably by the bDNA method.