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T Meyer

Publications and source records attributed to T Meyer.

At least 271 records · Page 15Linked to original sources
PubMed1997

Control of action potential-induced Ca2+ signaling in the soma of hippocampal neurons by Ca2+ release from intracellular stores.

Stimulus-induced increases in neuronal Ca2+ concentration are important signaling events for transcriptional regulation and neuronal plasticity. Electrical inputs are thought to mediate Ca2+ responses in the soma by triggering action potentials, which in turn open voltage-gated Ca2+ channels in the somatic plasma membrane. It is not yet known to what extent internal Ca2+ amplification contributes to the somatic Ca2+ responses. Here we used fluorescent Ca2+ measurements in cultured hippocampal neurons and report that the amplitude of the somatic Ca2+ increase triggered by field stimulation is independent of the extracellular Ca2+ concentration as long as the concentration is greater than 50 microM. Furthermore, significantly more La3+ has to be added extracellularly for blocking Ca2+ responses, as predicted from the reported La3+ dependence of voltage-gated Ca2+ channels. These measurements suggest that field stimulation-induced somatic Ca2+ responses in hippocampal neurons are largely attributable to Ca2+ release from intracellular stores. Only a small number of Ca2+ ions have to enter across the plasma membrane for this intracellular Ca2+ amplification process to occur. Rapid fluorescence-imaging measurements showed that the internal Ca2+ amplification occurs over 10-15 msec and linearly increases intracellular Ca2+ concentrations for up to 40 action potentials. At a fixed number of field pulses, frequencies of 40 Hz were optimal for somatic Ca2+ increases. Our studies suggest that the opening of intracellular Ca2+ release channels plays a crucial part in shaping the action potential-induced neuronal Ca2+ response.

Action Potentials↗
PubMed1997

Expression rate of vinculin isoforms in human aortocoronary saphenous vein grafts.

Aortocoronary bypass conduits derived from saphenous veins usually develop diffuse intimal thickening, one of the major causes of haemodynamically relevant graft stenosis. To elucidate the role of smooth muscle cell proliferation in late graft failure, specimens from highly stenotic or occluded vein grafts implanted into the arterial circulation for more than 5 years were tested for their expression rate of meta-vinculin. Since the cytoskeletal protein meta-vinculin is present exclusively in contractile smooth muscle cells, the determination of the relative amounts of meta-vinculin (150 kDa) and its low-molecular weight isoform vinculin (130 kDa) allows characterization of the phenotypic status of smooth muscle cells. Using immunoblotting techniques, the quantitative relation of meta-vinculin in tissue samples obtained from autoptic vein grafts (n=10) was measured and compared with those of native saphenous veins (n=6). In vein grafts, the fractional meta-vinculin content of the total vinculin immunoreactivity ranged from 32%-46% (mean 39.6%), whereas the range was 39%-53% (mean 46.7%) in native veins. By applying Student's t-test, a statistical significance was not demonstrated suggesting that the majority of smooth muscle cells in intimal thickenings consisted of a contractile phenotype. Immunohistochemically, the vinculin immunoreactivity in the intimal layer of vein grafts was reduced as compared to native saphenous veins. The distribution of vinculin in grafted veins closely resembled that in arteriosclerotic coronary arteries with intimal thickening. Hence, our biochemical data demonstrate parallels between the pathogenesis of late vein graft stenosis and degenerative arteriosclerotic lesions.

Actins↗
PubMed1997

Detection of impaired intrapulmonary convective mixing by aerosol bolus dispersion in patients with emphysema.

The broadening of an inhaled aerosol bolus (aerosol bolus dispersion) during respiration provides a noninvasive, nonradioactive, and easy to perform technique which is a measure of the extent of convective gas mixing processes in the lung and symmetry of ventilation during the breathing cycle. In this study, this technique was evaluated for the ability to detect ventilation inhomogeneities in pulmonary emphysema (PE). An aerosol bolus undergoes changes in shape between its inspiration and expiration. In comparison with the inhaled bolus, the exhaled bolus is more spread because of convective mixing and may have a shift in the location of the mode caused by asymmetries of filling and emptying of lung units. We hypothesized that in PE these changes differ from healthy subjects and are related to lung health. Twenty nine patients with PE inhaled 25 ml boluses containing 0.9 micron monodisperse aerosol particles into volumetric lung depths of 200, 400, 600, and 800 cm3. The control group were 79 healthy subjects. As each bolus was expired, we measured bolus dispersion (volumetric width at one-half aerosol concentration peak height) and mode shift. In patients with PE exhaled boluses were significantly broader than those exhaled by normal subjects at all volumetric lung depths. At a lung depth of 800 cm3, patients showed a bolus dispersion that was 52% greater than that of healthy subjects (p < 0.0001) and they exhaled the bolus mode earlier (p < 0.0001). Aerosol bolus parameters were not influenced by anthropometric data. Pulmonary function parameters indicating obstruction showed a weak negative correlation with aerosol bolus parameters (R2 < 0.4). The bolus parameters appeared to be supplemental to conventional lung function tests and significantly indicated marked ventilation asymmetry and altered convective gas transport in pulmonary emphysema. The results support the use of the aerosol bolus dispersion test in assessing peripheral parenchymal lung injury.

Administration, Inhalation↗
PubMed1997

Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.

Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, and eta have been expressed using the baculovirus expression system. The partially purified PKC subtypes have been studied for their substrate specificities and phospholipid-independent activation by various chemically different nontumor- and tumor-promoting agents, as well as their inhibition of kinase activity by staurosporine and two related compounds. An endogenous PKC-like kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition. Protamine sulfate was most efficiently phosphorylated by all of the PKC subtypes tested, although this phosphorylation was independent of phosphatidylserine (PS) and diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Except for PKC-zeta, all subtypes tested phosphorylated myelin basic protein (MBP), histone, or a peptide derived from the pseudosubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varying extents. Among the various agents tested, TPA most efficiently stimulated the kinase activities of the PKC subtypes in a phospholipid-dependent manner. Phorbol 12,13-dibutyrate (PDBu) was less effective than TPA but displayed no major difference among the subtypes. Activation of PKC-alpha by bryostatin-1 reached only half of the TPA response whereas the other subtypes were activated more effectively. The weak tumor promoter resiniferonol 9,13,14-orthophenyl acetate (ROPA) mainly stimulated PKC-alpha and PKC-gamma at 1 microM concentration, whereas PKC-epsilon and PKC-eta were much less activated. Sapintoxin D, mezerein, indolactam V, and resiniferatoxin at concentrations of 1-100 nM preferentially activated PKC-alpha in a DAG-like manner, whereas at 1 microM other subtypes were activated as well. Preferential activation of PKC-alpha was also noted for tinyatoxin and thapsigargin, but their mode of activation is unclear because these two compounds did not compete for the phorbol ester binding of the PKC subtypes as the other agents did. Of the three PKC inhibitors tested, staurosporine most efficiently inhibited kinase activity of the PKC subtypes, whereas K252a and CGP 41251 were at least 10 times less effective. However, K252a showed certain specificity for inhibition of PKC-alpha, and CGP 41251 failed to inhibit PKC-epsilon and PKC-zeta. Given the different substrate specificities and modes of activation by various tumor-promoting and nontumor-promoting agents, as well as the different sensitivities towards different inhibitors, our results indicate a divergence of individual PKC subtypes in signal transduction.

Animals↗
PubMed1997

Genomic organization of the human excitatory amino acid transporter gene GLT-1.

We present the genomic structure of the human glutamate transporter GLT-1 coding region, the intronic sequences adjacent to the exons, and oligonucleotide primer sequences for single strand conformational analysis. The exon-intron boundaries were determined using long-distance PCR and direct sequencing. The human GLT-1 coding region is composed of 10 exons spanning > 50 kb of genomic DNA. The exons range from 127 to 251 bp in length. The intron lengths vary considerably from 2.2 kb to > 15 kb. These data provide the basis for implementing a comprehensive screen for genetic alterations in the human GLT-1 gene using genomic DNA as a template.

ATP-Binding Cassette Transporters↗
PubMed1997

[Molecular biology findings in amyotrophic lateral sclerosis].

At presently, the etiology and pathogenesis of amyotrophic lateral sclerosis (ALS) are unknown. In recent years, the genetic background of hereditary motor neuron diseases has been partly defined. In particular, these advances represent an opportunity to improve our understanding of the pathogenesis of the familial and sporadic forms of ALS and thus provide a basis for rational therapeutic approaches. In this article, recent findings on the pathogenesis of the familial form of ALS and their implications for the sporadic form are discussed.

Amyotrophic Lateral Sclerosis↗
PubMed1997

Southern analysis of BT-R1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis.

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue.

Animals↗
PubMed1997

Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells.

Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes.

Animals↗
PubMed1997

Rapid down regulation of Ca2+ signals induced by endothelin-1 in a human bronchial epithelial cell line.

Ca2+ transients evoked by endothelin-1 (ET-1) were measured in single cells of an immortalized human tracheal epithelial cell line using Fura-2. ET-induced Ca2+ transients were compared to signals evoked via established phospholipase-C linked receptors (H1 histamine; P2y purinergic, ATP). Saturating concentrations of histamine (100 microM) and ATP (10 microM) caused Ca2+ transients of identical amplitude, whereas a saturating concentration of ET-1 (10 nM) on average resulted in a slightly smaller change in fluorescence ratio (80 +/- 27%). H1 and P2y induced Ca2+ signals caused by brief (10-30 s) application of the agonists were highly reproducible. No desensitization to these ligands was observed, if between two exposures cells were superfused with agonist-free solution for > or = 200 s. A single exposure to ET-1 (10 nM) for > or = 6 s reduced sensitivity of the cell to a second exposure to ET-1. On average, the signal upon a second application of 10 nM ET-1 had an amplitude of 30% of the first ET-1 induced signal in that cell. After two 10 s exposures to the peptide, less than 10% of the initial amplitude was measured. This desensitization did not affect responsiveness to histamine or ATP. No recovery from desensitization to ET-1 was observed for 12 h after a single brief treatment with the peptide. Thereafter, responsiveness to ET-1 re-appeared with a half time of about 5 h and was complete by about 20 h. Ca2+ signals to all three agonists were absent in thapsigargin treated cells. Their amplitude was not affected by superfusion of the cells with Ca(2+)-free solution. Under conditions of store-depletion either by ET-1 or by thapsigargin, a change from Ca(2+)-free to Ca(2+)-containing solution induced a slow rise in [Ca2+]i, suggesting the existence of a capacitative Ca2+ entry pathway. ET-receptors most likely of the ETA subtype are subject to a novel type of desensitization-at least with regard to Ca2+ signalling-which might reflect a signal transduction mechanism specific to ET receptors.

Adenosine Triphosphate↗
PubMed1997

Access to emergency care under TennCare: do patients understand the system?

STUDY OBJECTIVE: To determine patient understanding of how to properly gain access to urgent and emergency medical care under TennCare, a government-mandated managed health care initiative designed to replace Medicaid in Tennessee. METHODS: We prospectively surveyed a convenience sample of ED patients at university hospital ED with an annual census of 50,000 during two periods (summer 1994 and summer 1995). In 1994, 250 TennCare patients were enrolled (part 1). In 1995, 199 were enrolled (part 2). RESULTS: Patients from seven different TennCare managed care organizations (MCOs) were interviewed. Thirty-eight percent of part 1 patients and 37% of part 2 patients did not have or did not know the names of their primary care physicians (PCPs). Fifty-eight percent of the part 1 patients who knew their PCPs' names had never visited them. This figure had decreased to 25% by the time part 2 patients were surveyed. Seventy-three percent of part 1 patients interviewed did not call their PCPs before coming to the ED. This figure had decreased to 48% by the time part 2 patients were interviewed. Thirty-two percent of part 1 patients were aware that they were supposed to contact the PCP before visiting the ED, whereas 94% of part 2 patients were aware of this requirement. Thirty-one percent of part 1 patients and 40% of part 2 patients who tried to contact their PCPs were unsuccessful, most often because of a delay on the part of PCPs in returning calls. Fifty-six percent of part 1 patients and 69% of part 2 patients did not know that they might be held responsible for the bill if an ED visit was not considered a true emergency and was not approved by the MCO. CONCLUSION: Improvements in communication of pertinent information must be implemented in managed care systems such as TennCare to better inform participants of the proper use of the system. MCOs will not reduce inappropriate use of the ED if patients are not aware of their responsibilities and do not know their PCPs or how to gain access to them. PCP responsiveness to patients must also be improved.

Adult↗
PubMed1997

Attitudes toward the use of a metal detector in an urban emergency department.

STUDY OBJECTIVE: To determine the attitudes of patients, their family and friends, and ED staff toward a walk-through metal detector in the ED. METHODS: We conducted a survey of a convenience sample of ED patrons (patients and their friends and family) and staff at a university-affiliated Level I trauma center. RESULTS: We surveyed 176 patrons and 95 employees (35 nurses, 30 physicians, 16 security officers, and 14 staff members). Overall, 80% of the patrons and 85% of the employees said they liked the metal detector. Eighty-nine percent of the patrons and 73% of the employees said the metal detector made them feel safer. Only 12% of the patrons and 10% of the employees said the metal detector invaded their privacy or the privacy of others. Fewer than 1% of the patrons said they were less likely to return to our ED because of the metal detector, and 39% said it made them more likely to return. We detected no significant differences with regard to age, sex, or race. CONCLUSION: Most patrons and staff liked the metal detector and said it created a safer ED environment. Only a few disliked the presence of the metal detector or said it invaded their privacy. Institutions concerned about their employees' and patrons' perceptions of safety should consider installing metal detectors in their EDs.

Adolescent↗
PubMed1997

Glucocorticoid-dependent transcriptional repression of the osteocalcin gene by competitive binding at the TATA box.

The human osteocalcin gene is transcriptionally repressed by glucocorticoids. A specific binding element for the glucocorticoid receptor (GR) overlapping the TATA box of the human osteocalcin promoter has previously been identified. In the present study, the function of this element has been further characterized by competitive gel mobility-shift assay and transfection experiments. The GR and TATA-binding protein (TBP) bound to the cognate overlapping elements in a mutually exclusive manner. The GR preferentially inhibited the binding of TBP. The isolated DNA-binding domain of the GR is sufficient to compete for TBP binding. The integrity of both half-sites of the glucocorticoid response element (GRE) is required to effectively compete for TBP binding, and competitive binding of the GR is dependent on dimerization. Transient overexpression of TBP overrides the transcriptional repression of the osteocalcin promoter by glucocorticoids. We conclude that the repressive effect of glucocorticoids on this promoter is the result of competitive DNA binding to a basal transcriptional element and that it does not appear to require direct protein-protein interaction between the competitive factors.

Base Sequence↗
PubMed1997

Expression of myogenic marker proteins in human leiomyosarcoma.

A series of formalin-fixed, paraffin-embedded leiomyosarcomas (n = 11) was studied immunohistochemically for their expression of various myogenic marker proteins. According to their predominant histological appearance, the tumors were classified as well (n = 4), moderately (n = 5), or poorly (n = 2) differentiated. Using monoclonal anti-muscle specific actin antibodies from clone HHF35 all examined tumors were positively stained. Desmin was not always found in leiomyosarcomas, since positive staining could be demonstrated only in eight cases. As revealed by staining with anti-vinculin antibodies from clone hVIN-1 using the APAAP technique, all leiomyosarcomas with the exception of one expressed vinculin. Typically, the vinculin immunoreactivity was detected diffusely throughout the majority of neoplastic cells as well as in vascular smooth muscle cells of blood vessels. Nine leiomyosarcomas displayed a positive staining for calponin, an actin-binding protein expressed in smooth muscle cells and their precursors. The distribution of calponin resembled that of vinculin in decorating myofibrils of nearly all tumor cells. Actinin immunoreactivity was present in tumor cells of all cases, but was expressed also in nontumor cells such as epithelia. These results suggest that the monoclonal antibodies against vinculin and calponin may serve as additional diagnostic markers for myogenic differentiation in leiomyosarcomas and related tumors.

Actins↗
PubMed1997

Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate.

The beta2-adrenergic receptor (beta2AR) is prototypic of the large family of G protein-coupled receptors (GPCRs) whose desensitization and resensitization are regulated by intracellular kinases, arrestin proteins, phosphatases, and ill-defined components of the cellular endocytic machinery. The study of beta2AR signal transduction and behavior in living cells is technically difficult because of the relatively low cellular expression of the receptor and a lack of useful biological reagents. Availability of a functional beta2AR tagged with the highly sensitive Green Fluorescent Protein (GFP) could allow measurements of the various properties of the beta2AR. We demonstrate that a fully functional beta2AR/GFP can be engineered. In mammalian cells, beta2AR/S65T/GFP demonstrates strong, diffuse plasma membrane fluorescence when observed with 480 nm excitation. The fluorescent receptor binds agonist and antagonist, stimulates adenylyl cyclase, undergoes phosphorylation, and is internalized in a manner indistinguishable from wild-type receptor. We then show that its internal trafficking and surface mobility can be determined by measuring only the endogenous fluorescence of the conjugate. beta2AR/S65T/GFP was found to be localized on endosomal membranes in living cells within minutes of agonist treatment, and within 15 min it is observed in more complicated structures formed from fusion of multiple endosomes. Finally, its free diffusion (diffusion coefficient, 4.0-12 x 10(-9) cm2/sec) was assessed on living cells using photobleaching recovery measurements. This approach and the fidelity of the biochemical properties of the beta2AR/S65T/GFP demonstrate that real-time optical measurements of beta2AR (as well as other GPCR) interactions and dynamics on living cells are feasible.

Adenylyl Cyclases↗
PubMed1997

Synthesis and biological evaluation of monoindolyl and indolocarbazolyl oxazolones and imidazolones.

Eight compounds structurally related to protein kinase C inhibitor MDL 27032 and substituted with indole moieties were synthesized. Their activities towards protein kinase C (PKC) and protein kinase A (PKA) were determined. Their effect on PKC-mediated contraction of rat tracheal smooth muscle, their antiproliferative activity on two murine tumor cell lines, melanoma B16 and leukemia P388 and their antimicrobial activity on a gram-positive bacterium Bacillus cereus were also examined. The mammalian and bacterial cell antiproliferative activity, as well as vasorelaxant effect, observed for some of them could not be correlated to PKC or PKA inhibition. Only bulky bis-indolyl compounds exhibited biological activity in these experiments. Rigid indolocarbazoles had the strongest antiproliferative activity.

Animals↗
PubMed1997

Immunohistochemical detection of vinculin in human rhabdomyosarcomas.

The microfilament-associated protein vinculin is a major constituent of muscle tissue localized in costameres and Z-discs of the sarcomeric apparatus, where it is thought to play a pivotal role in the alignment of sarcomeric myofibrils and the transduction of mechanical force between the internal contractile machinery and the extracellular environment. In order to investigate whether anti-vinculin antibodies are helpful in confirming the commitment of rhabdomyosarcomas to the myogenic pathway, we studied immunohistochemically the expression pattern of vinculin in a series of 7 human rhabdomyosarcomas including those of embryonal, botryoid, and pleomorphic subtypes. Using monoclonal antibody from clone hVIN-1 by APAAP techniques on formalin-fixed, paraffin-embedded tissue, all but one tumor, which was a primitive embryonal rhabdomyosarcoma, demonstrated a significant positive vinculin staining. Vinculin expression was most prominent in differentiated tumors with a focal staining pattern showing a high degree of correlation with rhabdomyoblasts, whereas a diffuse staining was observed in areas in which small, poorly differentiated tumor cells alone were present. Since vinculin immunoreactivity could also be demonstrated in cases of leiomyosarcoma, the positive immunohistochemical detection of vinculin was not exclusively restricted to mesenchymal tumors derived from sarcomeric muscle tissue. Immunodetectable amounts of nebulin could be revealed only in two embryonal rhabdomyosarcomas. Our results suggested that the positive identification of rhabdomyosarcoma achieved by using antibodies against vinculin in addition to other known myogenic markers may be particularly useful in the differential diagnosis of anaplastic, poorly differentiated sarcomas.

Adult↗
PubMed1997

No mechanical role for vinculin in strain transduction in primary bovine osteoblasts.

Vinculin is thought to play a central role in linking the actin cytoskeleton to the integrin adhesion proteins of focal contacts and also in cell-cell adhesion sites. We have investigated aspects of attachment and the transduction of mechanical stimulation on the function of vinculin and its assembly in primary bovine osteoblasts. The dynamic attachment process was concomitant with the reassembly of vinculin from a soluble cytoplasmic pool into Triton-insoluble focal adhesions. Immunoblotting experiments demonstrated a slight increase in the amount of vinculin concentrated in focal contacts that was paralleled by a discrete decrease in the Triton-soluble fraction of cytoplasmic vinculin. Sixty cycles of 1 Hz deformations at 0.02% (hypophysiological, no change in cell division rate), 0.2% (within the normal physiological range), and 1% (hyperphysiological) resulted in a rapid and reversible disassembly of vinculin from focal contacts to a homogeneous cytoplasmic localization, although alterations in the shape and morphology of cells could not be detected. Although all mechanical loading protocols dramatically depleted vinculin from focal contacts, its initial distribution in adhesion plaques was fully restored within the next 60 min, demonstrating the highly dynamic and reversible shift of this protein from a membrane-associated pool to the cytoplasm. By first depleting vinculin from the focal adhesion sites by applying 0.02% strain, waiting 60 s, and then applying 0.2% strain, we show that the transduction of mechanical deformation was identical with controls that had not been pretreated. This indicates that vinculin does not play a role in mechanical transduction, that significant mechanical forces are not transmitted through vinculin, and also that loss of vinculin at the focal adhesion site does not have significant effects, in the short term, on cell adhesion.

Animals↗