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T Hartmann

Publications and source records attributed to T Hartmann.

At least 91 records · Page 5Linked to original sources
PubMed1996

Genes contributing to Alzheimer's disease.

We propose that Alzheimer's disease (AD) is a single disease with a common metabolic APP-beta A4-amyloid pathway. The multiple genetic and other factors already identified to induce this pathway are reviewed. The molecular genetics of AD has been successfully studied within the last years, and we now can account for the genetic and molecular alterations underlying the majority of familial AD cases inherited with an autosomal dominant pattern of complete penetrance. AD in these pedigrees can be caused by missense mutations within the recently identified PS1 (S182) gene on chromosome 14 (AD3 locus) and the PS2 (STM2/E5-1) gene on chromosome 1, in addition to previously described point mutations of the beta A4-amyloid protein precursor (APP) gene on chromosome 21 (AD1 locus). The majority of AD cases, however, appears to be sporadic or 'familial' in terms of an increased family-associated AD-probability. Genetic risk factors contributing to AD in these cases have also been identified. On chromosome 19, allelic segregation of the APOE gene with both late onset 'familial' (AD2) and sporadic AD has been demonstrated, with the APOE epsilon 4 allele conferring a relatively higher risk of developing AD at an earlier age. Several other risk factors have also been proposed, including the alpha 1-antichymotrypsin allele A (ACT-A), the 5-repeat allele of the VLDL-receptor (VLDL-R) gene, the A2 allele of the HLA-A locus, and possibly yet unknown mitochondrial mutations. All these findings are discussed against the background of what is known about APP metabolism leading to beta A4 amyloid formation, a process that is also modified by APP expression level, alternative splicing of APP exon 15, extracellular signalling and intracellular sorting.

Alzheimer Disease↗
PubMed1995

Thrombin receptor polymorphism in Chinese hamster.

We report the existence of a new Chinese hamster thrombin receptor allele, characterized by an in-frame insertion of three nucleotides at position 250 of the published sequence. As a consequence, an additional proline is inserted into a proline-rich region of the extracellular amino-terminal domain of the receptor. A corresponding proline at this position is also found in the rat thrombin receptor. A silent base-pair change is found in the cytoplasmic tail of the receptor gene. Single-strand conformation polymorphism and sequence analysis indicate this new receptor allele is present in several cell lines derived from different individual Chinese hamsters. Embryonic CHEF IIC9 cells and primary culture cells are homozygous for this new allele. In contrast, the CCL39 lung fibroblast cell line is heterozygous for both the new and old alleles. Both alleles are transcribed into mRNA and code for functional receptors. Given the allelic distribution and sequence alignment with thrombin receptors from other species, we propose that the new sequence represents the actual predominant allele in Chinese hamster.

Alleles↗
PubMed1995

Tryptase, the dominant secretory granular protein in human mast cells, is a potent mitogen for cultured dog tracheal smooth muscle cells.

Hyperplasia of airway smooth muscle cells is present in the airways of asthmatic patients and may contribute to the development of the bronchial hyperresponsiveness that occurs in these patients. Because tryptase is an abundant component of mast cell granules and has demonstrated growth-stimulatory effects in other mesenchymal cells (J. Clin. Invest. 1991; 88:493-499), the goal of our study was to determine whether tryptase is a mitogen for airway smooth muscle cells. The mitogenic effects of tryptase were tested in passages 1 through 5 of dog tracheal smooth muscle cells, either by counting smooth muscle cells or by monitoring uptake of bromodeoxyuridine (BrdU) into cellular DNA during S-phase. With respect to its efficacy, at a near maximal concentration (4 nM), tryptase increased cell numbers 2.1 +/- 0.2- or 2.8 +/- 0.6-fold above controls after 2 or 4 days, respectively, and these increases were approximately the same as those induced by platelet-derived growth factor (50 ng/ml) or 10% calf serum. With respect to potency, tryptase caused concentration-dependent increases in BrdU uptake, as detected in an enzyme-linked immunosorbent assay or by counting BrdU-labeled nuclei, with an EC50 of 2 nM. Pretreatment of tryptase with diisopropylfluorophosphate, to reduce markedly its catalytic as a activity as a proteinase, attenuated its growth-stimulated effects by 58 +/- 16%. Tryptase-induced mitogenesis was not a nonspecific effect of all serine proteinases, because thrombin, another proteinase with mitogenicity for fibroblasts, stimulated neither increases in cell counts nor BrdU uptake in our cells. We conclude that tryptase is a potent mitogen for airway smooth muscle cells in culture.

Animals↗
PubMed1994

Soluble endothelium-associated adhesion molecules in patients with Graves' disease.

The targeting and recruitment of inflammatory cells to vascular endothelium in Graves' disease (GD) is mediated by intercellular adhesion molecule-1 (ICAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), and vascular cell adhesion molecule-1 (VCAM-1). We have studied serum levels of soluble ICAM-1 (sICAM-1), soluble ELAM-1 (sELAM-1), and soluble VCAM-1 (sVCAM-1) in patients with GD (n = 21) and in patients with iodine-deficient goitre (IDG) (n = 23). The serum levels of sICAM-1 were markedly elevated in patients with GD before treatment with thiamazole (median 560 ng/ml versus 185 ng/ml in patients with IDG). In addition, elevated serum concentrations of sELAM-1 (median 85 ng/ml versus 33 ng/ml, respectively) and sVCAM-1 (median 42 ng/ml versus 15 ng/ml, respectively) were observed in patients with GD (P < 0.01 for all). The serum levels of sELAM-1 and sVCAM-1 dropped significantly after initiation of therapy and were within the normal range after 4, and 8 weeks of therapy, respectively. Serum levels of sICAM-1 were elevated even after 8 weeks of therapy. Serum levels of sVACM-1 and sICAM-1 correlated with the serum concentrations of anti-thyroid-stimulating hormone (TSH)-receptor antibodies (TSHR-R) (n = 21; r = 0.929 and r = 0.810, respectively) and anti-thyroid peroxidase antibodies (TPO-Ab) (n = 21; r = 0.673 and r = 0.750, respectively). However, no correlation between sELAM-1 and TPO-Ab, TSHR-R, and anti-thyroglobulin antibodies (Tg-Ab), respectively, could be found. In addition to thyroid hormones and autoantibodies, serum concentrations of sELAM-1 and sVCAM-1, but not sICAM-1, could be useful as clinical markers for disease activity.

Autoantibodies↗
PubMed1994

Modulation of thrombin and thrombin receptor peptide mitogenicity by human lung mast cell tryptase.

In previous studies, mast cell tryptase acted as a potent mitogen for fibroblasts from human lung and rodent embryonic tissue but failed to stimulate growth of cultured rat aortic vascular smooth muscle cells (VSMC). The current study shows that tryptase inhibits DNA synthesis in VSMC stimulated by thrombin. However, it does not affect the stimulation of DNA synthesis by the synthetic thrombin receptor peptide Ser-Phe-Phe-Leu-Arg-Asn-Pro (SFFLRNP), which mimics the amino-terminus of thrombin receptor proteolytically activated by thrombin. Nor does tryptase alter the mitogenic response of VSMC to purified growth factors, such as platelet-derived growth factor (PDGF). These data suggest that tryptase inhibits thrombin-induced DNA synthesis without interfering with intracellular mitogenic signaling pathways activated by thrombin or other growth factors. This study further suggests that tryptase neither cleaves nor inactivates thrombin. Therefore, inhibition of thrombin's mitogenic effects by tryptase is not mediated by destruction of thrombin itself. The inhibition by tryptase of thrombin-induced DNA synthesis in VSMC contrasts with the stimulatory effect of tryptase on fibroblasts, in which synergy is observed with thrombin, with thrombin receptor peptide and with other growth factors. These data provide in vitro evidence that mast cell tryptase interferes with thrombin-stimulated vascular smooth muscle growth and suggest that tryptase is a multifunctional growth factor whose actions are cell specific.

Animals↗
PubMed1994

[Apolipoprotein E and Alzheimer dementia. Personal results and brief literature review].

The apolipoprotein E allele (Apo-E gene) status was determined in 147 individuals from a longitudinal study. 53 satisfied NINCDS-ADRDA criteria for probable or possible Alzheimer's disease (AD), 37 were non-demented healthy controls. 31 patients with AD had one or two Apo-E 4 alleles (phenotypes 1 x 4-2; 20 x 4-3; 10 x 4-4) compared with only 9 controls (8 x 4-3; 1 x 4-4). Within the AD group, patients homozygous for Apo-E 4 had an earlier onset of illness (7 of 10 cases were presenile) and more severe cognitive impairment at the time of examination after similar durations of illness as patients without Apo-E 4. This may reflect an influence of the Apo-E 4 gene dosage on the onset and course of illness, which is probably independent of chromosome 14 mutations.

Aged↗
PubMed1992

Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation.

Previously we have shown that the COOH-terminal 100 residues (A4CT) of the amyloid protein precursor (APP), which carry the sequence of the amyloid beta A4 protein of Alzheimer's disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte lysate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Müller-Hill, B., Masters, C. L., and Beyreuther, K. (1988) EMBO J. 7, 949-957). Here we report that aggregation of this COOH-terminal APP fragment A4CT and also of beta A4 itself depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT and beta A4 in the wheat germ expression system resulted in only monomeric forms. We have identified the factors which are capable of transforming both soluble A4CT and beta A4 into insoluble and aggregating molecules. Monomeric A4CT or beta A4 expressed in the wheat germ lysate could be transformed into aggregating molecules by the addition of metal-catalyzed oxidation systems. The addition of radical scavengers such as ascorbic acid, trolox, and amino acids prevented the aggregation process induced by the radical initiators. Thus, the aggregation of amyloidogenic APP fragments if analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis requires amino acid oxidation and protein cross-linking induced by radical generation systems.

Amyloid beta-Protein Precursor↗
PubMed1992

Construction and performance of a rapid scan monochromator for multiwavelength fluorimetry.

A rapid scan monochromator was constructed for multiwavelength illumination of biological samples. A light weight holographic grating (2400 grooves/mm) was driven by a rapid stepper motor with 0.014 degrees angular resolution in an f/4 monochromator. Angular velocity and acceleration kinetics were optimized to minimize the time for grating rotation and stabilization. The times required for shifting output wavelengths by 20, 100, and 300 nm were 6, 7, and 9 ms, respectively. Wavelength selection and dwell times were software controlled and reproducible over extended data collections. Instrument utility was demonstrated by measurement of intracellular calcium in fura-2-labeled Chinese hamster lung fibroblasts at excitation wavelengths of 340, 360, and 380 nm. Compared to available filter wheel and chopper devices, the rapid scan monochromator has advantages of rapid and software-selectable wavelength control, excellent optical alignment, small size, and low cost. Applications of this device include illumination optics for multiwavelength microfluorimetry and scanning absorption spectroscopy, and detection optics for microspectroscopy.

Animals↗
PubMed1992

Hormonal regulation of Cl transport in polar airway epithelia measured by a fluorescent indicator.

Cl transport mechanisms in polarized cultures of canine tracheal epithelium were examined using an Ussing-type chamber with independent mucosal and serosal perfusion. Cl activity was monitored continuously from fluorescence of entrapped 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). When added to the serosal (but not mucosal) solution, isoproterenol increased Cl fluxes across the apical membrane Cl more than fourfold. Apical Cl transport was sensitive to diphenylamine-2-carboxylate (DPC) but not to furosemide, whereas basolateral membrane Cl transport was sensitive to furosemide but not to DPC. Based on a mathematical model of Cl transport, we developed a sensitive protocol to measure hormone-sensitive Cl transport. In Cl-loaded cells in which basolateral Cl transport was partially inhibited by furosemide, mucosal Cl removal caused no Cl efflux before but rapid efflux (0.25 mM/s) after addition of isoproterenol or chlorophenylthio-cAMP. In the presence of indomethacin to block prostaglandin production, elevation of intracellular Ca by bradykinin or 4-bromo-A23187 did not cause Cl efflux, nor did Ca buffering with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid affect stimulation by the cAMP pathway. Phorbol 12-myristate 13-acetate increased Cl efflux submaximally (0.09 mM/s) but did not affect maximal stimulation by cAMP agonists. Methoxamine did not alter apical or basolateral membrane Cl transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Calcium-dependent regulation of Cl secretion in tracheal epithelium.

In primary cultures of dog tracheal epithelium, isoproterenol produced a transient increase in short-circuit current (Isc) (duration 30 s; maximal increase, 32 +/- 5 microA/cm2). This was followed by a more slowly developing sustained increase (9 +/- 3 microA/cm2), which mimicked the response to N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP). The transient and sustained responses had dissociation constants for isoproterenol of 2 x 10(-8) and 2 x 10(-9) M, respectively. Bradykinin (in the presence of indomethacin), substance P, histamine, and thrombin produced only transient increases in Isc. The time courses of these transients closely paralleled changes in concentration of intracellular Ca ([Ca2+]i) as measured with fura 2. For different mediators, there was a significant correlation between the maximal transient increase in Isc and the maximal increase in [Ca2+]i. The transients in Isc were not associated with elevation of adenosine 3',5'-cyclic monophosphate (cAMP) and were unaffected by pretreatment with DBcAMP, which abolishes the steady-state increase in response to isoproterenol. Both the transient increases in Isc and [Ca2+]i were inhibited by pretreatment with the Ca chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate abolished the transient increases in [Ca2+]i and Isc in response to isoproterenol but not to bradykinin. These results provide evidence that 1) isoproterenol and bradykinin elevate [Ca2+]i by different mechanisms, and 2) Ca elevation is associated with a transient increase in Isc, whereas increased cAMP is associated with a smaller sustained increase.

Animals↗
PubMed1992

Human tryptase as a potent, cell-specific mitogen: role of signaling pathways in synergistic responses.

Mast cells are hypothesized to participate in processes leading to tissue fibrosis in human lung and skin. To explore the possible involvement of mast cell mediators in fibrogenesis, the mitogenic activity of mast cell tryptase from human lung was examined in vitro. The results indicate that human tryptase is a potent inducer of DNA synthesis in fibroblasts from multiple sources, including human lung. As demonstrated by mitogenic responses in fibroblasts, but not in vascular smooth muscle cells, tryptase is a mitogen with target cell specificity. Additionally, specificity is demonstrated by the differences in mitogenic activity of tryptase in comparison with thrombin, a structurally related mitogenic proteinase. Examination of the mitogenic effects of tryptase in the presence of other mitogens reveals synergy with mitogens that act through receptors coupled to intrinsic tyrosine kinases (insulin, epidermal growth factor, and basic fibroblast growth factor) or to G proteins (thrombin and serotonin). In the latter case, studies in Chinese hamster lung fibroblasts using specific receptor agonists and antagonists or receptor-transfected cell lines reveal a requirement for the activation of a G protein (Gi) negatively coupled to adenylate cyclase to act synergistically with tryptase. These data establish that human tryptase is a potent and specific mitogen in vitro and suggest that mitogenic signals generated by tryptase can interact synergistically with signals generated by both tyrosine kinase-coupled and G protein-coupled growth factor receptors.

Carbachol↗
PubMed1991

Interfacial energies and surface-tension forces involved in the preparation of thin, flat crystals of biological macromolecules for high-resolution electron microscopy.

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1 degree, and perhaps less than 0.2 degrees, over areas as large as 1 micron2 or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.

Adsorption↗
PubMed1991

Mast cell tryptase is a mitogen for cultured fibroblasts.

Mast cells appear to promote fibroblast proliferation, presumably through secretion of growth factors, although the molecular mechanisms underlying this mitogenic potential have not been explained fully by known mast cell-derived mediators. We report here that tryptase, a trypsin-like serine proteinase of mast cell secretory granules, is a potent mitogen for fibroblasts in vitro. Nanomolar concentrations of dog tryptase strongly stimulate thymidine incorporation in Chinese hamster lung and Rat-1 fibroblasts and increase cell density in both subconfluent and confluent cultures of these cell lines. Tryptase-induced cell proliferation appears proteinase-specific, as this response is not mimicked by pancreatic trypsin or mast cell chymase. In addition, low levels of tryptase markedly potentiate DNA synthesis stimulated by epidermal growth factor, basic fibroblast growth factor, or insulin. Inhibitors of catalytic activity decrease the mitogenic capacity of tryptase, suggesting, though not proving, the participation of the catalytic site in cell activation by tryptase. Differences in Ca++ mobilization and sensitivity to pertussis toxin suggest that tryptase and thrombin activate distinct signal transduction pathways in fibroblasts. These data implicate mast cell tryptase as a potent, previously unrecognized fibroblast growth factor, and may provide a molecular link between mast cell activation and fibrosis.

Amino Acid Sequence↗
PubMed1990

Strong and persistent activation of inositol lipid breakdown induces early mitogenic events but not Go to S phase progression in hamster fibroblasts. Comparison of thrombin and carbachol action in cells expressing M1 muscarinic acetylcholine receptors.

In order to evaluate the role of phosphoinositide turnover in growth factor action, we expressed human M1 muscarinic acetylcholine (Hm1) receptors in Chinese hamster lung fibroblasts (CCL39 cell line). In the transfected cells (39M1-81 clone), but not in wild type fibroblasts, the muscarinic agonist carbachol induced a release of inositol phosphates as strong as alpha-thrombin, a very potent growth factor and activator of phosphoinositide-specific phospholipase C (PLC) in this cell system. In contrast to thrombin, carbachol-stimulated PLC activity was not inhibited by pertussis toxin treatment of cells. At concentrations that elicited a comparable initial rate of inositol phosphate release (10 nM for thrombin and 0.1 mM for carbachol), both agents gave rise to an identical calcium signal and equally stimulated Na+/H+ exchange and the transcription of the early genes c-jun, c-fos, and c-myc. Surprisingly, however, carbachol is not a mitogen for 39M1-81 cells, and even if tested in association with insulin or fibroblast growth factor, its effects on cell proliferation remained weak when compared with thrombin. Also, the muscarinic agonist did not stimulate soft agar colony forming capacity and did not prevent growth arrest in Go upon serum deprivation of cycling 39M1-81 cells. The failure of carbachol to induce cell proliferation could not be attributed to rapid and complete desensitization of Hm1 receptors nor to the activation of inhibitory pathways like adenylyl cyclase stimulation. We conclude that strong and persistent activation of phosphoinositide turnover elicits early biochemical events generally associated with mitogenesis, but is not sufficient to stimulate or maintain continuous cell proliferation. On the basis of our results, we postulate that thrombin mitogenesis depends critically on signaling events different from phosphoinositide turnover, possibly the stimulation of a receptor tyrosine kinase or a Gi protein-activated tyrosine kinase.

Adenylate Cyclase Toxin↗
PubMed1990

Model of ion transport regulation in chloride-secreting airway epithelial cells. Integrated description of electrical, chemical, and fluorescence measurements.

An electrokinetic model was developed to calculate the time course of electrical parameters, ion fluxes, and intracellular ion activities for experiments performed in airway epithelial cells. Model variables included cell [Na], [K], [Cl], volume, and membrane potentials. The model contained apical membrane Cl, Na, and K conductances, basolateral membrane K conductance, Na/K/2 Cl and Na/Cl symport, and 3 Na/2 K ATPase, and a paracellular conductance. Transporter permeabilities and ion saturabilities were determined from reported ion flux data and membrane potentials in intact canine trachea. Without additional assumptions, the model predicted accurately the measured short-circuit current (Isc), cellular conductances, voltage-divider ratios, open-circuit potentials, and the time course of cell ion composition in ion substitution experiments. The model was used to examine quantitatively: (a) the effect of transport inhibitors on Isc and membrane potentials, (b) the dual role of apical Cl and basolateral K conductance in cell secretion, (c) whether the basolateral symporter requires K, and (d) the regulation of apical Cl conductance by cAMP and Ca-dependent signaling pathways. Model predictions gave improved understanding of the interrelations among transporting systems and in many cases gave surprising predictions that were not obvious without a detailed model. The model developed here has direct application to secretory or absorptive epithelial cells in the kidney thick ascending limb, cornea, sweat duct, and intestine in normal and pathophysiological states such as cystic fibrosis and cholera.

Animals↗