Tyrosinase gene variants in different rabbit strains.
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Animals↗
Biomedical subjects
M Müller
Publications and source records attributed to M Müller.
Alveolar epithelial type II cell apoptosis in vivo during resolution of keratinocyte growth factor-induced hyperplasia in the rat.
Keratinocyte growth factor (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF/kg body weight or diluent. Lungs were fixed 1, 2, 3, 5, and 7 days later. Apoptosis was identified by TdT-mediated dUTP nick-end labeling (TUNEL), double-labeling for TUNEL and the type II cell marker MNF116, and electron microscopy. Fas, FasL, Bax, Bcl-2, and pro- and active caspase-3 were studied by immunohistochemistry. Changes were quantified by stereology. Cell type specificity was investigated by immunofluorescence double staining. Type II cells exhibited Fas, FasL, Bcl-2, and procaspase-3 irrespective of treatment and time. Immunoelectron microscopy revealed Fas at the apical type II cell membrane. Bax staining was prominent in controls (45-95% of type II cell surface fraction), markedly decreased during hyperplasia at days 2 (20-40%) and 3 (0-10%), and reappeared at day 7 (25-45%) when apoptosis was prominent. Remnants of apoptotic type II cells were incorporated in membrane-bound vacuoles of type II cell neighbors as well as alveolar macrophages. The results indicate that type II cells can enter the Fas/FasL/caspase-3 pathway regulated by Bax and Bcl-2. High Bcl-2:Bax levels favor type II cell survival and a low rate of apoptosis during hyperplasia. Low Bcl-2:Bax levels favor type II cell apoptosis during resolution. Because of time-dependent changes that occur within a short time, the KGF-treated rat lung provides a useful in vivo model to investigate apoptosis in the context of tissue remodeling and repair.
Animals↗
Functional assessment of chest wall integrity after methylmethacrylate reconstruction.
BACKGROUND: All patients with extensive resection of the anterolateral chest wall and the sternum followed by reconstruction with methylmethacrylate substitutes were assessed prospectively 6 months after the operation to delineate chest wall integrity with pulmonary function and cine-magnetic resonance imaging. METHODS: Twenty-six patients underwent chest wall reconstruction by use of methylmethacrylate between 1994 and 1998 due to primary tumors in 35%, metastases in 27%, T3 lung cancer in 19%, and debridement for radionecrosis and osteomyelitis in 19% of patients. Three to eight ribs were resected and additional sternum resection was performed in 39% of patients. RESULTS: There was no 30-day mortality. All patients were extubated after the operation without need for reintubation. Prosthesis dislocation occurred in 1 patient and infection in 2 patients during follow-up. Nineteen patients (73%) suffered no restrictions of daily activities. Clinical examination revealed normal shoulder girdle function in 77% of patients. There was no significant difference between preoperative and postoperative FEV1 (forced expiratory volume in 1 second) measurements in patients with lobectomy or wedge resections. Cinemagnetic resonance imaging revealed concordant chest wall movements during respiration in 92% of patients without paradoxical movements or implant dislocations being observed. CONCLUSIONS: Large defects of the anterolateral chest wall and sternum can be reconstructed efficiently with methylmethacrylate substitutes with minimal morbidity and excellent cosmetic and functional outcome.
Adult↗
Orientation of the infrared transition moments for an alpha-helix.
Appropriate values for the orientation of the amide transition dipoles are essential to the growing use of isotopically edited vibrational spectroscopy generally in structural biology and to infrared dichroism measurements on membrane-associated alpha-helices, in particular. The orientations of the transition moments for the amide vibrations of an alpha-helix have been determined from the ratio of intensities of the A- and E(1)-symmetry modes in the infrared spectra of poly(gamma-methyl-L-glutamate)(x)-co-(gamma-n-octadecyl-L-glutamate)( y) oriented on silicon substrates. Samples possessing a high degree of alignment were used to facilitate band fitting. Consistent results were obtained from both attenuated total reflection and transmission experiments with polarized radiation, yielding values of Theta(I) = 38 degrees, Theta(II) = 73 degrees, and Theta(A) = 29 degrees, relative to the helix axis, for the amide I, amide II, and amide A bands, respectively. The measurements are discussed both in the context of the somewhat divergent older determinations, and in relation to the helix geometry and results on model amide compounds, to resolve current uncertainties in the literature.
Biophysical Phenomena↗
Transgenic rat hearts expressing a human cardiac troponin T deletion reveal diastolic dysfunction and ventricular arrhythmias.
OBJECTIVE: Familial hypertrophic cardiomyopathy (FHC) due to mutations of cardiac troponin T (cTnT) is associated with a high frequency of sudden death even in the absence of cardiac hypertrophy. To investigate the causal relationship of cTnT mutations and this particular phenotype, we sought to establish a transgenic rat model for the disease. METHODS: Transgenic rats were generated expressing human wild-type cTnT or two truncated cTnT molecules (del ex16, del ex15/16), resulting from an intron 15 splice donor site mutation previously observed in FHC patients. Transgenic rat hearts were characterized by histology, immunohistochemistry and in the 'working heart'. RESULTS: Human wild-type and del ex16 cTnT were stably expressed and incorporated into the sarcomere of transgenic cardiomyocytes. Del ex16 transgenic rats revealed a lower level of expression (4-5%) than human wt cTnT animals (25-40%). In the 'working heart' model del ex16 hearts exhibited significant systolic and diastolic dysfunction without cardiac hypertrophy. In contrast, human wt cTnT hearts showed improved contractile performance and moderate myocardial hypertrophy. After 6 months of daily physical exercise one del ex16 rat died suddenly and three out of five del ex16 hearts revealed ventricular tachycardia/fibrillation. No arrhythmia was observed in human wt cTnT expressors. Myofibrillar disarray was present in del ex16 hearts after training but not in human wild-type cTnT rats or non-transgenic controls. CONCLUSION: A human cTnT deletion overexpressed in transgenic rats exerts a dominant-negative effect and mimics the phenotype of FHC with diastolic dysfunction and arrhythmias. By contrast, human cTnT wild-type animals reveal a gain of function and cardiac hypertrophy without arrhythmias.
Animals↗
Oxygen radical generation of neutrophils: a reason for oxidative stress during marathon running?
Hematological parameters and blood markers that indicate oxidative stress, such as lipid peroxides (LPO), reduced and oxidized glutathione (GSH, GSSG), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were measured in 18 marathon runners before, immediately after the race, and after 8 days of rest. In parallel, the oxygen radical generation of neutrophils (PMN) was measured by chemiluminescence in six randomly selected runners. After the race, a 4.4-fold enhanced PMN count and a 1.4-fold increased capacity to generate oxygen radicals of the PMN (2.20+/-0.38 vs. 3.12+/-0.69 arb. unit/10(6) cells) were found. Consequently, a 6.25-fold increased capacity to generate oxygen radicals of the post-run blood (7.26+/-1.3 vs. 45.40+/-10.3 arb. unit/ml blood) was calculated. This points to PMN as an important oxygen radical source established in the runners' blood, which could contribute to the oxidative stress indicated in the post-run blood by increased LPO (11.46+/-3.09 vs. 13.09+/-3.14 micromol/l plasma), GSSG (0.038+/-0.003 vs. 0.045+/-0. 005 mmol/l blood) and GSSG/GSH ratio (3.8+/-0.5 vs. 4.1+/-0.6%) and by decreased SOD (15.63+/-1.78 vs. 14.58+/-1.51 10(3)U/mmol Hb) and GSH-Px (485.1+/-107.1 vs. 434.9+/-101.7 U/mmol Hb). Despite the decline of the oxygen radical source during rest, the oxidative stress in the blood did not decrease in all runners.
Adult↗
Telomerase activity and expression of telomerase subunits in the testicular tissue of infertile patients.
OBJECTIVE: Determination of telomerase activity and the expression of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in the testicular tissue of patients with infertility arising from various causes. DESIGN: Prospective observational study. SETTING: A university hospital. PATIENT(S): Thirty-three patients with azoospermia arising from various causes. There were 12 testicular biopsy specimens from patients with Sertoli cell-only syndrome, 9 from patients with maturation arrest, and 12 from patients with obstructive azoospermia and normal histologic findings. INTERVENTION(S): Thirty-three testicular biopsies. MAIN OUTCOME MEASURE(S): Correlation of histologic findings at testicular biopsy with telomerase activity, hTERT, and hTR. RESULT(S): All 12 biopsy specimens from patients with obstructive azoospermia were positive for telomerase activity, hTR, and hTERT. Biopsy specimens from the 9 patients with maturation arrest were positive for telomerase activity in 8 cases, hTR in 9 cases, and hTERT in 5 cases. None of the patients with Sertoli cell-only syndrome showed either telomerase activity or hTERT, but all of them showed hTR. CONCLUSION(S): Telomerase activity and evidence of hTERT in testicular tissue are highly sensitive and highly specific markers of gametogenesis, which could gain in importance as part of the fertility workup before microinjection procedures.
DNA-Binding Proteins↗
Paracrine effects of angiotensin-converting-enzyme- and angiotensin-II-receptor- inhibition on transcapillary glucose transport in humans.
The paracrine renin-angiotensin-system (RAS) is increasingly recognized to play an important role in the regulation of both, regional vascular tone and regional glucose metabolism. To date, however, a selective investigation of paracrine RAS effects in an in vivo clinical setting was beyond technical reach. We here set out to selectively study the metabolic effects of paracrine RAS inhibition at different levels in healthy volunteers (n = 8). For this purpose bradykinin, enalaprilate and losartan were administered locally to the interstitial space fluid in skeletal muscle by means of reverse microdialysis and transcapillary glucose transport was measured simultaneously. During reverse microdialysis with bradykinin and enalaprilate a significant decrease in arterial-interstitial-gradient for glucose (AIG(glu)) was observed (from 1.49 +/- 0.08 mM to 0.12 +/- 0.63 mM (p = 0.018) for bradykinin and from 1.5 +/- 0.07 mM to 0.24 +/- 0.67 mM (p = 0.043) for enalaprilate). In contrast, losartan had no effect on AIG(glu). The changes in transcapillary glucose transport during bradykinin and enalaprilate administration were accompanied by significant increases in interstitial lactate levels which was most pronounced for bradykinin (from 0.14 +/- 0.01 mM to 0.40 +/- 0.07 mM, p = 0.018). We conclude that paracrine angiotensin-converting-enzyme (ACE) inhibition but not angiotensin II (AT-II) receptor blockade decreases AIG(glu) and facilitates transcapillary glucose transport due to an increase in interstitial bradykinin concentration. These results support the concept that blood pressure control with ACE-inhibitors but not with AT-II-receptor-antagonists has beneficial long term metabolic consequences in hypertensive, hyperinsulinemic subjects.
Adult↗
A mitochondrion in Entamoeba histolytica?
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Animals↗
Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae.
Yeast vacuoles are highly dynamic and flexible organelles. In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for sample preparation that so far have been barely successful in yeast. Cells of leucine-limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase. To relieve leucine-limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid-based expression of a leucine marker gene. Such cultures reached more than two-fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.
Animals↗
Effects of chloride transport inhibition and chloride substitution on neuron function and on hypoxic spreading-depression-like depolarization in rat hippocampal slices.
Chloride fluxes play a crucial role in synaptic inhibition, cell pH regulation, as well as in cell volume control. In many neuropathological processes, cell swelling is a pivotal parameter, since cell volume changes and the dimension of the interstitial space critically modulate synchronized neuronal activity as well as the tissue's susceptibility to seizures or spreading depression. This study therefore focuses on the effects of different Cl(-) transport inhibitors and Cl(-) substitution on neuronal function and hypoxia-induced changes in rat hippocampal tissue slices. Orthodromically evoked focal excitatory postsynaptic potentials were depressed by furosemide (2mM), 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (1mM) and Cl(-) substitution by methylsulfate, but were enhanced by 4,4'-dinitrostilbene-2,2'-disulfonic acid (1mM). All four treatments induced multiple population spike firing in response to single orthodromic volleys, suggesting reduced synaptic inhibition. Antidromic population spikes increased following Cl(-) withdrawal, were unaffected in the presence of furosemide and 4, 4'-dinitrostilbene-2,2'-disulfonic acid, but were abolished by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The amplitude of the hypoxic spreading-depression-like extracellular potential shift was reduced by furosemide, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and Cl(-) withdrawal, i.e. by the same treatments that depressed orthodromically evoked postsynaptic potentials. Furosemide prolonged the time to onset and the duration of the spreading-depression-like extracellular potential shift, while 4, 4'-dinitrostilbene-2,2'-disulfonic acid shortened the time to onset. Spreading-depression-related cell swelling was recorded as the shrinkage of relative interstitial space, which was measured as tetramethylammonium-chloride space. Neither the Cl(-) transport inhibitors nor Cl(-) withdrawal had any detectable effect on spreading-depression-related cell swelling. CA1 pyramidal neurons usually hyperpolarized during drug application and their input resistance decreased. Cl(-) withdrawal increased their input resistance and caused spontaneous burst firing. Hypoxia caused the expected spreading-depression-like rapid, near complete depolarization of single pyramidal neurons and drastically reduced their input resistance. The three Cl(-) transport inhibitors and Cl(-) withdrawal delayed the onset of the hypoxic depolarization. In low Cl(-) solutions, the apparent threshold potential at which spreading depression was triggered shifted to more positive membrane potentials. The final voltage of the hypoxic depolarization was, however, not affected. It appears from these results that the reduction in the hypoxic spreading-depression-like extracellular potential shifts by Cl(-) transport inhibitors is at least partially attributable to desynchronization of depolarization, not to decreased depolarization in individual cells. Other contributing factors could be changes in recording conditions, depression of swelling-induced amino acid release from glial cells and unspecific side-effects of the applied drugs. Desynchronization could also account for the delayed spreading-depression onset. It is concluded that Cl(-) fluxes play a role in the triggering of spreading depression, but the spreading-depression-like depolarization itself or its self-regenerative character is not mediated by Cl(-).
Animals↗
Function and regulation of ATP-binding cassette transport proteins involved in hepatobiliary transport.
Hepatobiliary transport of endogenous and exogenous compounds is mediated by the coordinated action of multiple transport systems present at the sinusoidal (basolateral) and canalicular (apical) membrane domains of hepatocytes. During the last few years many of these transporters have been cloned and functionally characterized. In addition, the molecular bases of several forms of cholestatic liver disease have been defined. Combined, this has greatly expanded our understanding of the normal physiology of bile formation, the pathophysiology of intrahepatic cholestasis, as well as of drug elimination and disposition processes. In this review recent advances, with respect to function and regulation of ATP binding cassette transport proteins expressed in liver, are summarized and discussed.
ATP-Binding Cassette Transporters↗
Assessment of middle cerebral artery diameter after aneurysmal subarachnoid hemorrhage by transcranial color-coded duplex sonography.
This study examined if vasospasm after aneurysmal subarachnoid hemorrhage could be visualized by middle cerebral artery (MCA) diameter changes in transcranial color-coded duplex sonography (TCCS). Comparative measurements between mean blood velocity (MBV) and MCA diameter were carried out in 17 patients in 76 instances. At two depth ranges (proximal, 60 55 mm: distal, 50-45 mm) two observers assessed the MCA diameter as indicated by the visualized blood flow column. At both points of measurement, the diameter differences between the two observers were within the ¿ 2 S.D. range of the mean difference indicating interobserver agreement. In 17 instances, MBV was > 120 cm/s indicating vasospasm but MBV did not correlate with absolute or relative diameter changes. MCA diameter assessment in TCCS seems reproducible. Because TCCS imaging is influenced by several factors comparative angiographic studies are necessary to clarify the TCCS findings.
Female↗
Partial impairment of cytokine responses in Tyk2-deficient mice.
To assess the role of the Janus kinase (Jak) family member Tyk2, we have generated Tyk2-/- mice. In contrast to other Jaks, where inactivation leads to a complete loss of the respective cytokine receptor signal, Tyk2-/- mice display reduced responses to IFNalpha/beta and IL-12 and a selective deficiency in Stat3 activation in these pathways. Unexpectedly, IFNgamma signaling is also impaired in Tyk2-/- mice. Tyk2-/- macrophages fail to produce nitric oxide upon lipopolysaccharide induction. Tyk2-/- mice are unable to clear vaccinia virus and show a reduced T cell response after LCMV challenge. These data imply a selective contribution of Tyk2 to the signals triggered by various biological stimuli and cytokine receptors.
Animals↗
Specific gain- and loss-of-function phenotypes induced by satellite-specific DNA-binding drugs fed to Drosophila melanogaster.
DNA-binding pyrrole-imidazole compounds were synthesized that target different Drosophila melanogaster satellites. Compound P31 specifically binds the GAGAA satellite V, and P9 targets the AT-rich satellites I and III. Remarkably, these drugs, when fed to developing Drosophila flies, caused gain- or loss-of-function phenotypes. While polyamide P9 (not P31) suppressed PEV of white-mottled flies (increased gene expression), P31 (not P9) mediated three well-defined, homeotic transformations (loss-of-function) exclusively in brown-dominant flies. Both phenomena are explained at the molecular level by chromatin opening (increased accessibility) of the targeted DNA satellites. Chromatin opening of satellite III by P9 is proposed to suppress PEV of white-mottled flies, whereas chromatin opening of satellite V by P31 is proposed to create an inopportune "sink" for the GAGA factor (GAF).
AT Rich Sequence↗
Origins of hydrogenosomes and mitochondria.
Complete genome sequences for many oxygen-respiring mitochondria, as well as for some bacteria, leave no doubt that mitochondria are descendants of alpha-proteobacteria, a finding for which the endosymbiont hypothesis can easily account. Yet a wealth of data indicate that mitochondria and hydrogenosomes - the ATP-producing organelles of many anaerobic protists - share a common ancestry, a finding that traditional formulations of the endosymbiont hypothesis less readily accommodates. Available evidence suggests that a more in-depth understanding of the origins of eukaryotes and their organelles will hinge upon data from the genomes of protists that synthesize ATP without the need for oxygen.
Aerobiosis↗
Immunization against human papillomavirus infection and associated neoplasia.
BACKGROUND: Chimeric virus like particles (CVLPs) constructed by fusing human papillomavirus type 16 (HPV16) E7 sequences into the C-terminus of the viral L1 gene constitute the first generation of preventive and therapeutic HPV vaccines. Even though vaccination with DNA is highly efficient in the induction of a cytotoxic T-cell (CTL) response utilization of a DNA vaccine in the HPV context, it has been hampered by concern for the oncogenic potential of the E6 and E7 proteins encoded by the viral oncogenes. OBJECTIVE: To consider the use and impact of E7 DNA for immunization. EXPERIMENTAL: In addition to hemagglutination inhibition, a versatile assay to measure neutralization of yeast cell-derived pseudovirions carrying a green fluorescence reporter gene has now been developed. Mice immunized with the HPV16 CVLPs generate E7-specific CTLs, which kill E7 expressing or E7 peptide loaded RMA-cells, protect against tumor formation by syngeneic HPV transformed cells and also induce regression of already established tumors. Since generation of CTL response is achieved by presentation of epitopes as short peptides together with appropriate MHC class I molecules, complete proteins are not required. Instead a shuffled E7 protein has now been used successfully for generating CTL responses comparable to the CVLP responses in mice. CONCLUSIONS: Our preliminary results suggest that immunization with E7 shuffled DNA yields a response directed against the authentic E7 protein. Furthermore, booster immunization with E7 shuffled DNA would avoid inhibition by neutralizing antibodies, however, further studies are needed to guarantee that the shuffled E7 protein lacks oncogenic activity.
Animals↗
Assessment of genetic relatedness of vaginal isolates of Candida albicans from different geographical origins.
PCR fingerprinting with single non-specific primers was used to type vaginal isolates of C. albicans from Portugal, Angola, Madagascar, and two regions of Germany (Berlin and Munich). In addition to analysing isolates that exhibited the normal biotype of C. albicans, the study included atypical strains that failed to assimilate glucosamine and N-acetylglucosamine, which were isolated from women in Angola and Madagascar. A total of 212 strains of C. albicans were studied, representing 87 different multi-locus genotypes. The genotypes of strains from each geographical population were highly similar but not identical. There was one exception: a strain from Portugal grouped with the typical strains from Angola. The typical and especially the atypical populations from Africa displayed less genotype variation than the populations from Europe. The Portuguese samples exhibited the greatest genotypic heterogeneity. Distance analysis (UPGMA) revealed a statistically weak correlation between genotype and geographical origin of the C. albicans isolates.
Adolescent↗