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PubMed2006

Dielectric measurement of individual microtubules using the electroorientation method.

Little is known about the electrostatic/dynamic properties of microtubules, which are considered to underlie their electrostatic interactions with various proteins such as motor proteins, microtubule-associated proteins, and microtubules themselves (lateral association of microtubules). To measure the dielectric properties of microtubules, we developed an experiment system in which the electroorientation of microtubules was observed under a dark-field microscope. Upon application of an alternating electric field (0.5-1.9 x 10(5) V/m, 10 kHz-3 MHz), the microtubules were oriented parallel to the field line in a few seconds because of the dipole moment induced along their long axes. The process of this orientation was analyzed based on a dielectric ellipsoid model, and the conductivity and dielectric constant of each microtubule were calculated. The analyses revealed that the microtubules were highly conductive, which is consistent with the counterion polarization model-counterions bound to highly negatively charged microtubules can move along the long axis, and this mobility might be the origin of the high conductivity. Our experiment system provides a useful tool to quantitatively evaluate the polyelectrolyte nature of microtubules, thus paving the way for future studies aiming to understand the physicochemical mechanism underlying the electrostatic interactions of microtubules with various proteins.

Electric Impedance↗

PubMed1987

[Evaluation of work systems in the textile industry in relation to the development of pathological changes in the genital organs of seamstresses].

The investigations which have proceeded for some years in clothing plants of the Western Pomeranian region with the aim of finding out pathological changes involving female machine operators' genital organs, have revealed a number of unfavourable phenomena, mainly, psychoneurotic changes in the initial phase and pelvic congestion syndrome (pcs), as described by Taylor, in a later phase. The paper is aimed at evaluation of one factor possibly affecting workers' health, i.e. work systems. The data collected from the plant managers, female chargehands and machine operators themselves, as well as from our own observations and investigations, revealed that the test groups of women were performing a piece-job, direct-line and belt-system production. For eight hours a day they performed strictly defined labour requiring great precision in course of repeated tasks in predetermined time limits. A more relaxed system, so called "direct line-synchro nests", arranged temporarily on experimental basis, appeared less efficient and was finally given up for economical reasons. Altogether, 575 machine operators have been examined. The control group consisted of 100 women working in the so called work-gang system offering a greater freedom of movements (cleaners, cutters, store- keepers). The obtained results are presented in two tables. The rate of pcs was found to be statistically significantly higher in machine operators, as compared to controls.

Ergonomics↗

PubMed1996

Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF.

The gene therapy approach presented in this protocol employs a polymer encapsulated, xenogenic, transfected cell line to release human ciliary neurotrophic factor (hCNTF) for the treatment of Amyotrophic Lateral Sclerosis (ALS). A tethered device, containing around 10(6) genetically modified cells surrounded by a semipermeable membrane, is implanted intrathecally; it provides for slow continuous release of hCNTF at a rate of 0.25 to 1.0 micrograms/24 hours. The semipermeable membrane prevents immunologic rejection of the cells and interposes a physical, virally impermeable barrier between cells and host. Moreover, the device and the cells it contains may be retrieved in the event of side effects. A vector containing the human CNTF gene was transfected into a line of baby hamster kidney cells (BHK) with calcium phosphate using a dihydrofolate reductase-based selection vector with a SV40 promoter and contains a HSV-tk killer gene. hCNTF is a potent neurotrophic factor which may have utility for the treatment of ALS. Systemic delivery of hCNTF in humans has been frustrated by peripheral side effects, the molecule's short half life, and its inability to cross the blood-brain barrier. The gene therapy approach described in this protocol is expected to mitigate such difficulties by local intrathecal delivery of a known quantity of continuously-synthesized hCNTF from a retrievable implant.

Amyotrophic Lateral Sclerosis↗

PubMed2006

Validation of three-dimensional cardiac image integration: use of integrated CT image into electroanatomic mapping system to perform catheter ablation of atrial fibrillation.

INTRODUCTION: Accurate visualization of the complex left atrial (LA) anatomy and the location of an ablation catheter within the chamber is important in the success and safety of ablation for atrial fibrillation (AF). We describe the integration of CT into an electroanatomic mapping (EAM) system and its validation in patients undergoing catheter ablation for AF. METHODS AND RESULTS: Thirty patients (59.2 +/- 8 years, 25 M) with paroxysmal (12) and persistent (18) AF underwent ablation using CT image integration into an electroanatomic mapping system. CT registration using the pulmonary veins as markers (landmark) was achieved with an error of 6.4 +/- 2.8 mm with repeat registration required in two patients. Registration of the CT by best fit to a electroanatomic geometry (surface) was achieved with an error of 2.3 +/- 0.4 mm. There was no significant difference in the regional LA registration error at superior (1.7 +/- 0.7 mm), inferior (2.2 +/- 1.4 mm), septal (1.7 +/- 0.8 mm), and lateral (1.7 +/- 0.7 mm, P = 0.13) sites. Cardiac rhythm at the time of CT did not have a significant effect on total or regional surface registration accuracy (mean total 2.5 +/- 0.3 in AF patients vs 2.3 +/- 0.5 in SR patients, P = 0.22). The integrated CT was used to guide the encirclement of the pulmonary veins (PV) in pairs with electrical isolation achieved by maintaining ablation along the ablation line in 58 of 60 PV pairs. Postprocedural PV angiography did not demonstrate significant stenosis. CONCLUSION: CT image integration into an EAM system was successfully performed in patients undergoing catheter ablation for AF. With a greater appreciation of the complex and variable nature of the PV and LA anatomy this new technology may improve the efficacy and safety of the procedure.

Atrial Fibrillation↗

PubMed2003

The role of retinoic acid in the morphogenesis of the neural tube.

We have examined the role of the signalling molecule, retinoic acid, in the process of neurulation and the subsequent growth and differentiation of the central nervous system using quail embryos that have developed in the absence of retinoic acid. Such retinoic acid-free embryos undergo abnormal neural tube formation in terms of its shape and structure, but the embryos do not display spina bifida or exencephaly. The neural tubes have a wider floor plate, a thicker roof plate and a different dorsoventral shape. Phalloidin staining and electron microscopy revealed alterations in the actin filaments and the junctional complexes of the cell layer lining the lumen. Initially the neural tubes proliferated at the same rate as normal, but later the proliferation rate declined drastically and neuronal differentiation was highly deficient. There were very few motoneurons extending neurites into the periphery, and within the neural tube axon trajectories were chaotic. These results reveal several functions for retinoic acid in the morphogenesis and growth of the neural tube, many of which can be explained by defective notochord signalling, but they do not suggest that this molecule plays a role in neural tube closure.

Actin Cytoskeleton↗

PubMed2006

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures--a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study.

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid "raft" mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 degrees C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (lo+so) two-phase coexistence region below 8+/-2 degrees C at ambient pressure. With increasing temperature, a lo+ld+so three-phase region is formed, which extends up to approximately 27 degrees C, where a liquid-ordered/liquid-disordered (lo+ld) immiscibility region is formed. Finally, above 48+/-2 degrees C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, ld). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the lo+ld (+so) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 degrees C, and a pressure range, which extends up to about 2 kbar for T=37 degrees C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.

2-Naphthylamine↗

PubMed1996

Renal and neuronal abnormalities in mice lacking GDNF.

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for embryonic midbrain dopaminergic, spinal motor, cranial sensory, sympathetic, and hindbrain noradrenergic neurons, and is available to these cells in vivo. It is therefore considered a physiological trophic factor and a potential therapeutic agent for Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Here we show that at postnatal day 0 (P0), GDNF-deficient mice have deficits in dorsal root ganglion, sympathetic and nodose neurons, but not in hindbrain noradrenergic or midbrain dopaminergic neurons. These mice completely lack the enteric nervous system (ENS), ureters and kidneys. Thus GDNF is important for the development and/or survival of enteric, sympathetic and sensory neurons and the renal system, but is not essential for catecholaminergic neurons in the central nervous system (CNS).

Animals↗

PubMed1996

Thermal response in murine L929 cells lacking alpha B-crystallin expression and alpha B-crystallin expressing L929 transfectants.

We investigated the role of alpha B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45 degrees C induced thermotolerance in these cells to a heat challenge at 45 degrees C administered 24 h later. The thermotolerance ratio at 10(-1) isosurvival was 1.7. Expression of alpha B-crystallin gene was not detected during the 24 h incubation at 37 degrees C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected alpha B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with alpha B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive alpha B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of alpha B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected alpha B-crystallin can contribute to increased thermoresistance.

3T3 Cells↗

PubMed1996

Inhalation of prostacyclin (PGI2) for 8 hours does not produce signs of acute pulmonary toxicity in healthy lambs.

OBJECTIVE: To study the potential side effects and toxicity of inhaling prostacyclin (PGI2) aerosol for 8 h. DESIGN: In a prospective, randomized study 14 healthy lambs received either PGI2 (n = 7) or 0.9% NaCl (n = 7) as an aerosol for 8 h. SETTING: Institute for Surgical Research of the Ludwig-Maximilians-University of Munich. INTERVENTIONS: All animals were studied under general anesthesia in a prone position. They were first intubated endotracheally and later tracheotomized. PGI2 solution (median dose 28 ng/kg per min) or 0.9% NaCl was administered with a jet nebulizer (delivery rate 4-10 ml/h; mass median diameter of aerosol particles 3.1 microns). Bronchoalveolar lavage was performed before and after the inhalation period to collect epithelial lining fluid of alveoli. MEASUREMENTS AND RESULTS: Hemodynamic and respiratory parameters, systemic resorption (plasma levels of 6-keto-prostaglandin-F 1 alpha), in vitro bleeding time, collagen-induced platelet aggregation and global biochemical and cellular composition of the epithelial lining fluid were examined in order to assess the side effects and signs of acute pulmonary toxicity induced by inhaled PGI2. No statistically significant differences were found between the PGI2 and the control groups for any of the parameters examined. CONCLUSION: Inhalation of PGI2 (28 ng/kg per min) over a period of 8 h in healthy lambs does not produce major side effects or acute pulmonary toxicity.

Acute Disease↗

PubMed1991

Behavioural evidence for a peripheral component in the enhanced antinociceptive effect of a low dose of systemic morphine in carrageenin-induced hyperalgesic rats.

This study reinvestigated the possible contribution of a peripheral action of systemic morphine in the modulation of the response to noxious pressure on inflamed paws, using a supraspinally integrated test and various low doses of naloxone. Rats received an injection of carrageenin into the right hindpaw which resulted in an ipsilateral inflammatory response and decreased threshold to noxious pressure. Four hours post-carrageenin, the injection of 1 mg/kg i.v. morphine induced a significantly enhanced antinociceptive effect on the inflamed compared to the non-inflamed paws. Intrapantar injection of extremely low doses of naloxone (0.5 and 1 micrograms in a volume of 0.1 ml) significantly reduced this effect (naloxone being more effective when administered at the same time as morphine, compared to 15 min later), while equal doses of naloxone given systemically were inactive. These data confirm that synergism of peripheral and central actions may result in the augmented analgesic potency of morphine in rats subjected to inflammatory conditions. In addition, they provide further evidence for the complexity of opioid actions in inflammatory processes. In particular, the results are in line with the hypothesis that the paradoxical antinociceptive effect of extremely low doses of i.v. naloxone described in several studies is due to a central action.

Analgesia↗

PubMed1991

International EMS: lessons learned in Costa Rica.

Health care planners in Costa Rica, seeking to upgrade their emergency medical services, contracted with United States agencies for additional funding and established a national program for the development of an emergency medical service system. Three years later some of the problems and accomplishments of both the planning and early implementation phases have been identified and include the importance of a detailed assessment of current system components, resources, and priorities; the early implementation of an accurate data collection system; early planning for system self-sufficiency; the vital role of the lead agency with formal lines of authority; the careful development of local expertise and leadership. The continuing efforts in Costa Rica provide a valuable resource for other countries as they face their emergency health care needs.

Costa Rica↗

PubMed2004

Neurochemical and behavioral deficits consequent to expression of a dominant negative EphA5 receptor.

The Eph family tyrosine kinase receptors and their ligands have been linked to axon guidance and topographic mapping of the developing central nervous system. More specifically, the EphA5 receptor has been shown to play a role in development of hippocamposeptal, retinotectal and thalamocortical projections. Recently, a line of transgenic mice was developed which expresses a truncated EphA5 receptor lacking a functional tyrosine kinase domain. In a previous study, axonal tracing revealed that medial hippocampal axons in this strain projected laterally and ventrally away from their normal target area. In the current study, both transgenic and wild-type controls were evaluated in unconditioned (rotorod and locomotor activity) and conditioned (water maze and active avoidance) behavior tasks which tested hippocampal and striatal functioning. Compared to controls, the transgenic strain did not show differences in rotorod motor activity but did show a transient deficit in spatial navigation ability and a consistent impairment in active avoidance. The dominant-negative mutant receptor also resulted in a decrease in striatal dopamine and serotonin concentrations with no change in hippocampal monoamines. Collectively, these data suggest that animals expressing a truncated EphA5 receptor show deficits related to striatal functioning.

Animals↗

PubMed2001

Subdural engraftment of serotonergic neurons following spinal hemisection restores spinal serotonin, downregulates serotonin transporter, and increases BDNF tissue content in rat.

Spinal hemisection injury at T13 results in development of permanent mechanical allodynia and thermal hyperalgesia due to interruption and subsequent loss of descending inhibitory modulators such as serotonin (5-HT) and its transporter (5-HT(T)). We hypothesize that lumbar transplantation of non-mitotic cells that tonically secrete 5-HT and brain-derived neurotrophic factor (BDNF) will restore alterations in 5-HT and 5-HT(T) systems within the spinal dorsal horn. We used an immortalized rat neuronal cell line derived from E13 raphe (RN46A-B14) which is shown to secrete 5-HT and BDNF in vitro and in vivo. Three groups (n=35) of 30 day old male Sprague-Dawley rats were spinally hemisected at T13 and 28 days later received either lumbar RN46A-V1 control empty-vector (n=15) or RN46A-B14 (n=15) intrathecal grafts, or no transplant. Twenty-eight days following transplantation, animals were perfused and tissue examined for changes in 5-HT, 5-HT(T), and BDNF at the site of transplantation or at lumbar enlargements (L5). Immunohistochemistry revealed that RN46A-B14, but not RN46A-V1 cells, increased 5-HT tissue staining at L5 in the dorsal white matter as well as in superficial dorsal horn laminae I and II on both ipsilateral and contralateral sides, results confirmed by ELISA. Transplantation of RN46A-B14 cells significantly reduced ipsilateral 5-HT(T), upregulated after injury. Significantly increased levels of BDNF were also observed after RN46A-B14 transplantation but were not localized to particular spinal laminae. These results are consistent with recovery of locomotor function and reductions in chronic pain behaviors observed behaviorally after RN46A-B14 transplantation and supports the pragmatic application of cell-based therapies in correcting damaged circuitry after spinal cord injury.

Animals↗

PubMed2005

Development of an ischemic tolerance model in a PC12 cell line.

Although ischemic tolerance has been described in a variety of primary cell culture systems, no similar in vitro models have been reported with any cell line. A model of ischemic preconditioning in the rat pheochromocytoma PC12 cell line is described here. When compared to nonpreconditioned cells, preexposure of PC12 cells to 6 hours of oxygen and glucose deprivation (OGD) significantly increased cell viability after 15 hours of OGD 24 hours later. Flow cytometry analysis of cells labeled with specific markers for apoptosis, Annexin V, and Hoechst 33342, and of DNA content, revealed that apoptosis is involved in OGD-induced PC12 cell death and that preconditioning of the cells mainly counteracts the effect of apoptosis. Immunocytochemistry of caspase-3, a central executioner in the apoptotic process, further confirmed the activation of apoptotic pathways in OGD-induced PC12 cell death. This model may be useful to investigate the cellular mechanisms involved in neuronal transient tolerance following ischemia.

Animals↗

PubMed1988

Relationship of first primary palatine rugae to the maxillary canines in man.

Fifty dentate maxillary casts, obtained from thirty-four males and sixteen females, were mounted in the three-dimensional co-ordinate system of a contour meter. A transverse line of reference (x-axis) was drawn through the distal contact points of the maxillary canines, at a right angle to the y-axis which passed through the contact points of the maxillary central incisors. Relative to the canine-to-canine baseline, the locations of the lateral borders of the right and left first primary rugae were determined. The rugae were distributed on both the anterior and posterior sides of the baseline, and the anterior and posterior distances from the baseline were on the average about 1 mm. The age of the subjects, ranging from 12 to 52 years, appeared to be unrelated to the frequencies of anteriorly and posteriorly positioned rugae. Right and left first primary rugae, located on the anterior side of the baseline, showed a minute asymmetry in their topography. By contrast, there was topographical symmetry when the rugae were located on the posterior side of the baseline.

Adolescent↗

PubMed1989

Detection of virus-specific RNA-dependent RNA polymerase activity in extracts from cells infected with lymphocytic choriomeningitis virus: in vitro synthesis of full-length viral RNA species.

We have developed an in vitro assay for the lymphocytic choriomeningitis virus (LCMV) RNA-dependent RNA polymerase with ribonucleoprotein complexes extracted from acutely infected tissue culture cells. The RNA products synthesized in vitro corresponded in size to the full-length genomic L and S RNAs and subgenomic NP and GP mRNAs normally produced in vivo during acute LCMV infection. In a temporal analysis spanning the first 72 h of acute infection, the in vitro polymerase activity of ribonucleoprotein complexes was maximal at 16 h and declined significantly at later times. In contrast, the intracellular levels of the viral L protein (the putative polymerase protein) appeared to be maximal at 48 to 72 h postinfection. Our results suggest that the accumulation of L protein correlates with reduced viral replication and transcription at later times in acute infection and may be involved in the transition from acute to persistent LCMV infection.

Animals↗

PubMed1987

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

Cell Line↗

PubMed1999

Anterior cruciate ligament tears: MR imaging-based diagnosis in a pediatric population.

PURPOSE: To evaluate the diagnostic accuracy of primary and secondary magnetic resonance (MR) imaging findings of anterior cruciate ligament (ACL) tears in young patients with immature skeletal systems. MATERIALS AND METHODS: MR images obtained in 43 patients aged 5-16 years who underwent arthroscopy were retrospectively reviewed. Two reviewers evaluated primary findings (abnormal signal intensity, abnormal course as defined by Blumensaat angle, and discontinuity), secondary findings (bone bruise in lateral compartment, anterior tibial displacement, uncovering of posterior horn of lateral meniscus, posterior cruciate ligament line, and posterior cruciate angle), and meniscal and other ligamentous injuries. RESULTS: There were 19 ACL tears and 24 intact ACLs. Overall sensitivity and specificity of MR imaging in detecting ACL tears were 95% and 88%, respectively. Sensitivities of the primary findings were 94% for abnormal Blumensaat angle; 79%, abnormal signal intensity; and 21% discontinuity. The specificity of all primary findings was 88% or greater. The sensitivity and specificity of the secondary findings, respectively, were 68% and 88% for bone bruise; 63% and 92%, anterior tibial displacement; 42% and 96%, uncovered posterior horn of lateral meniscus; 68% and 92%, positive posterior cruciate line; and 74% and 71%, abnormal posterior cruciate angle. Fifteen (79%) patients had meniscal tears, and five (26%) had collateral ligament injuries. CONCLUSION: Primary and secondary findings of ACL tears in young patients have high specificity and are useful for diagnosis.

Adolescent↗