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Ralf Labugger

Publications and source records attributed to Ralf Labugger.

10 recordsLinked to original sources
PubMed2006

Age-associated cellular relocation of Sod 1 as a self-defense is a futile mechanism to prevent vascular aging.

Vascular aging is characterized by the presence of chronic oxidative stress. Although cytosolic Sod 1 has a key role in the detoxification of superoxide ((*)O(2)(-)), little is known about its importance in vascular aging. We found that inhibition of Sod 1 had no effect on (*)O2- generation. Furthermore, its expression decreased in an age-dependent manner. Interestingly, Sod 1 loses its membrane-association and is also lost from the caveolae with increasing age. Instead, a relocation of Sod 1 to the mitochondria takes place, presumably in an attempt to maintain mitochondrial integrity and to counter-balance age-associated oxidative stress. Unlike Sod 2, which is constitutively expressed in mitochondria to control (*)O2- radical fluxes, Sod 1 is not inactivated by peroxynitrite and is not nitrated as a function of age. These novel insights into oxidative stress-associated vascular aging and the understanding about how redox-systems are regulated in old age may identify new targets to ameliorate aging as the greatest cardiovascular risk factor.

Aging↗
PubMed2005

Fast and slow skeletal troponin I in serum from patients with various skeletal muscle disorders: a pilot study.

BACKGROUND: Detection of skeletal muscle injury is hampered by a lack of commercially available assays for serum markers specific for skeletal muscle; serum concentrations of skeletal troponin I (sTnI) could meet this need. Moreover, because sTnI exists in 2 isoforms, slow (ssTnI) and fast (fsTnI), corresponding to slow- and fast-twitch muscles, respectively, it could provide insight into differential injury/recovery of specific fiber types. The purpose of this study was to investigate whether the 2 isoforms of sTnI and their modified forms are present in the blood of patients with various skeletal muscle disorders. METHODS: Serial serum samples were obtained from 25 patients with various skeletal muscle injuries. Serum proteins were separated by a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol followed by Western blotting for sTnI with monoclonal antibodies specific to ssTnI and fsTnI. RESULTS: We observed (a) intact and, in some cases, degraded sTnI products; (b) evidence of posttranslational modifications in addition to proteolysis; and (c) differential detectability of both skeletal isoforms in the same patient. CONCLUSIONS: It is possible to monitor both sTnI isoforms; this could lead to the development of new diagnostic assays for skeletal muscle damage.

Antibodies, Monoclonal↗
PubMed2005

Expression and activity patterns of nitric oxide synthases and antioxidant enzymes reveal a substantial heterogeneity between cardiac and vascular aging in the rat.

We investigated the effects of aging and ischemia-reperfusion (I/R) injury on the expression and activity of nitric oxide (*NO) synthases and superoxide dismutase (SOD) isoforms. To this end we perfused excised hearts from young (6 months old) and old (31-34 months old) rats according to the Langendorff technique. The isolated hearts were, after baseline perfusion for 30 min, either subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion or were control-perfused (60 min normoxic perfusion). Both MnSOD and Cu,ZnSOD expression remained unchanged with increasing age and remained unaltered by I/R. However, SOD activity decreased from 7.55 +/- 0.1 U/mg protein in young hearts to 5.94 +/- 0.44 in old hearts (P<0.05). Furthermore, I/R led to a further decrease in enzyme activity (to 6.35 +/- 0.41 U/mg protein; P<0.05) in myocardium of young, but not in that of old animals. No changes in myocardial protein-bound 3-nitrotyrosine levels could be detected. Endothelial NOS (eNOS) expression and activity remained unchanged in aged left ventricles, irrespective of I/R injury. This was in steep contrast to peripheral (renal and femoral) arteries obtained from the same animals where a marked age-associated increase of eNOS protein expression could be demonstrated. Inducible NOS expression was undetectable either in the peripheral arteries or in the left ventricle, irrespective of age. In particular when associated with an acute pathology, which is furthermore limited to a certain time frame, changes in the aged myocardium with respect to enzymes crucially involved in maintaining the redox homeostasis, seem to be much less pronounced or even absent compared to the vascular aging process. This may point to heterogeneity in the molecular regulation of the cardiovascular aging process.

Aging↗
PubMed2004

Age-related changes of vitamin A status.

Ageing is an independent risk factor for the development of cardiovascular disease. The ageing process is known to be associated with increased oxidative stress and an increased risk for cardiovascular and other diseases, such as cancer. To delay this process, therapeutic strategies involving the use of naturally occurring antioxidants, such as vitamin A, have gained considerable interest. Therefore, we wanted to investigate in a model of mammalian ageing whether changes in tissue and plasma levels of vitamin A occur with increasing age. This would constitute a prime rationale for its dietary supplementation. Experiments were performed in three different age groups (4-6 months old, 19 months old, 32-35 months old) of F1 (F344 x BN) healthy male rats that were fed a normal diet without any additional supplementation. Vitamin A and carotenoids in plasma and major organs were measured by reverse-phase high-performance liquid chromatography. In 3-year-old rats, vitamin A levels were found to be decreased in plasma (P < 0.0001) as compared with young and middle-aged animals. However, they were markedly increased in the main storage organ (ie, the liver) (P < 0.01-0.0001), and also in the aortic vessel wall. They were undetectable in the heart, irrespective of age. Increased tissue levels of vitamin A, especially in the vasculature, may be part of an age-associated self-regulatory process of adaptation, possibly as a counter-regulation against oxidative tissue damage. Based upon the assumption that in elderly humans, as in our animal model, a similar demand-regulated mechanism may work independently of additional dietary vitamin A supplementation, one may question the strategy of large clinical interventional trials using vitamin A or its derivatives beyond normal dietary intake.

Aging↗
PubMed2003

[Molecular modifications of troponin I and T detected in serum from patients with acute myocardial infarction].

INTRODUCTION: Cardiac troponin I and T (cTnI and cTnT) are specific biochemical serum markers for acute myocardial infarction (AMI). However, cTnI diagnostic assays are plagued by difficulties, resulting in > 20-fold differences in measured values. These discrepancies may result from the release of the numerous cTnI modification products that are present in ischaemic myocardium. The resolution of these discrepancies requires an investigation of the exact forms of the troponins present in the bloodstream of patients after myocardial injury. MATERIAL AND METHODS: A Westernblot direct serum analysis protocol was developed that allowed us to detect intact cTnI and a spectrum of up to 11 modified products in the serum from patients with AMI. RESULTS: We document both a cTnI degradation pattern and the existence of phosphorylated cTnI in serum. The number and extent of these modifications reflect patterns similar to the time profiles of the routine clinical serum markers of total creatine kinase, creatine kinase-MB, and cTnI (determined by ELISA). Data from in vitro experiments, which were undertaken to study the degradation of human recombinant cTnI and cTnT when spiked in serum, indicate that some modification products present in patient serum existed in the myocardium. DISCUSSION: This pilot study defines, for the first time, what forms of cTnI and cTnT appear in the bloodstream of AMI patients, and it clarifies the lack of standardization between different cTnI diagnostic assays.

Biomarkers↗
PubMed2003

Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry.

BACKGROUND: Cardiac troponins are modified during ischemic injury and are found as a heterogeneous mixture in blood of patients with cardiovascular diseases. We present a strategy to isolate cardiac troponins from human biological material, by use of affinity chromatography, and to provide samples ready for direct analysis by mass spectrometry. METHODS: Cardiac troponins were isolated from human left ventricular tissue by affinity chromatography. Isolated troponins were either eluted and analyzed by Western blot or enzymatically digested while bound to affinity beads. The resulting peptide mixture was subjected to mass spectrometry for protein identification and characterization. The same method was used to analyze serum from patients with acute myocardial infarction (AMI). RESULTS: Affinity chromatography with antibodies specific for one cardiac troponin subunit facilitated the isolation of the entire cardiac troponin complex from myocardial tissue. The three different proteases used for enzymatic digestion increased the total protein amino acid sequence coverage by mass spectrometry for the three cardiac troponin subunits. Combined amino acid sequence coverage for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) was 54%, 48%, and 40%, respectively. To simulate matrix effects on the affinity chromatography-mass spectrometry approach, we diluted tissue homogenate in cardiac troponin-free serum. Sequence coverage in this case was 44%, 41%, and 19%, respectively. Finally, affinity chromatography-mass spectrometry analysis of AMI serum revealed the presence of cardiac troponins in a wide variety of its free and/or complexed subunits, including the binary cTnI-cTnC and cTnI-cTnC-cTnT complexes. CONCLUSIONS: Affinity chromatography-mass spectrometry allows the extraction and analysis of cardiac troponins from biological samples in their natural forms. We were, for the first time, able to directly confirm the presence of cardiac troponin complexes in human serum after AMI. This approach could assist in more personalized risk stratification as well as the search for reference materials for cardiac troponin diagnostics.

Amino Acid Sequence↗
PubMed2002

Cardiovascular aging is associated with vitamin E increase.

BACKGROUND: Aging is an independent risk factor for the development of cardiovascular disease. Therefore, therapies to delay vascular aging may have enormous medical consequences. In this context, vitamin E is of particular interest, mainly because of its antioxidative properties. METHODS AND RESULTS: In 3-year-old rats, which are not susceptible to atherosclerosis, vitamin E levels, as measured by reversed-phase high-performance liquid chromatography, were markedly increased both in plasma and in major organs (P<0.01 to P<0.0001). The highest increase (at least 70-fold) was found in the aortic wall. CONCLUSIONS: This unexpected accumulation of vitamin E appears to be a compensatory mechanism that attempts to counterbalance age-associated oxidative stress and that may represent a self-regulatory protective adaptation.

Adaptation, Physiological↗
PubMed2002

Solubilization, two-dimensional separation and detection of the cardiac myofilament protein troponin T.

Proteomics strongly relies on the separation of complex protein mixtures, with two-dimensional electrophoresis (2-DE) being a commonly used technique. However efficient separation requires adequate solubilization of the original sample which will determine whether all proteins are accurately represented. Cardiac muscle has presented a particular challenge to solubilization. Here we have optimized the solubilization, separation and detection of the myofilament protein troponin T (TnT). Human left ventricular tissue from a rejected donor transplant heart was homogenized under 19 different conditions and subjected to 2-DE and Western blot analysis for TnT. The optimal conditions for isoelectric focusing of intact TnT requires homogenization in 6 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, with the addition of NaCl (2.5 M final concentration). TnT degradation products present in this severely damaged heart however, were differentially extracted from both each other and the intact molecule under the various conditions used. Despite adequate focusing of TnT it was found that a nonglutaraldehyde silver staining protocol, that is compatible with subsequent mass spectrometry, has greatly reduced sensitivity for TnT compared to Coomassie blue. Thus, care is required to avoid misrepresentation of troponin T in proteomic analysis in cardiac tissue.

Blotting, Western↗
PubMedDate not supplied

Oxidative stress-associated vascular aging is independent of the protein kinase C/NAD(P)H oxidase pathway.

Aging is an independent risk factor for the development of cardiovascular disease. Vascular aging is mainly characterized by endothelial dysfunction, which, in turn, is primarily attributable to increased superoxide (O(2)(*)(-)) formation with age. To date, the source of this age-associated increased O(2)(*)(-) production remains obscure. We investigated whether like in hyperglycemia or hypertension protein kinase C (PKC)-mediated activation of the NAD(P)H oxidase system is involved. Here we show that both PKC translocation, necessary for its activation, and expression of the cytosolic subunits of the NAD(P)H oxidase, p47(phox) and p67(phox), remain unchanged with age. Therefore, we suggest that oxidative stress-associated vascular aging mechanistically differs from endothelial dysfunction seen in the context of other cardiovascular risk factors, for which the PKC/NAD(P)H oxidase pathway has been shown responsible.

Aging↗