Search PubMed⌕ Search

Biomedical subjects

G Link

Publications and source records attributed to G Link.

At least 19 recordsLinked to original sources
PubMed2002

The iron-loaded gerbil model revisited: effects of deferoxamine and deferiprone treatment.

Although the beneficial effects of deferoxamine (DFO) on iron-associated morbidity and mortality are well documented, the role of deferiprone (L1) in the management of transfusional iron overload is controversial. This debate involves not only the question of efficacy but also of safety, with particular emphasis on the risk of a paradoxical aggravation of iron toxicity by L1. We used the iron-loaded gerbil model introduced by Carthew et al to compare the chelating efficacy of L1, DFO, or both in two gerbil strains treated by means of weekly iron-dextran injections: Psammomys obesus and pathogen-free Mongolian gerbils (Meriones unguiculatus). The difference between the high mortality and advanced hepatocellular necrosis observed in iron-loaded P obesus and the absence of mortality and limited morbidity encountered in pathogen-free Mongolian gerbils is most likely explained by the prevention of coincidental laboratory infections in the latter group. Iron-chelating treatment in all experimental groups resulted in a significant decrease in hepatic iron concentrations and normalization of mitochondrial respiratory enzyme activities, with combined L1 and DFO treatment being the most efficient, followed, in decreasing order, by DFO and L1 as single-drug treatments. Judged by tissue iron concentrations, mitochondrial enzyme activity, and hepatic histology, we could find no evidence of a paradoxical aggravation of iron toxicity by L1 in either of the two series of studies. Although these data appear to be reassuring, the present controversy related to the role of L1 in the development of hepatic cirrhosis should be eventually settled by clinical studies evaluating the effects of long-term iron-chelating treatment.

Animals↗
PubMed2001

Structure of the P700(+ )A1(-) radical pair intermediate in photosystem I by high time resolution multifrequency electron paramagnetic resonance: analysis of quantum beat oscillations.

The geometry of the secondary radical pair P700(+)A1(-), in photosystem I (PSI) from the deuterated and 15N-substituted cyanobacterium Synechococcus lividus, has been determined by high time resolution electron paramagnetic resonance (EPR), performed at three different microwave frequencies. Structural information is extracted from light-induced quantum beats observed in the transverse magnetization of P700(+)A1(-) at early times after laser excitation. A computer analysis of the two-dimensional Q-band experiment provides the orientation of the various magnetic tensors of with respect to a magnetic reference frame. The orientation of the cofactors of the primary donor in the g-tensor system of is then evaluated by analyzing time-dependent X-band EPR spectra, extracted from a two-dimensional data set. Finally, the cofactor arrangement of P700(+)A1(-) in the photosynthetic membrane is deduced from angular-dependent W-band spectra, observed for a magnetically aligned sample. Thus, the orientation of the g-tensor of P700(+) with respect to a chlorophyll based reference system could be determined. The angle between the g1(z) axis and the chlorophyll plane normal is found to be 29 +/- 7 degrees, while the g1(y) axis lies in the chlorophyll plane. In addition, a complete structural model for the reduced quinone acceptor, A1(-), is evaluated. In this model, the quinone plane of is found to be inclined by 68 +/- 7 degrees relative to the membrane plane, while the P700(+)-A1(-) axis makes an angle of 35 +/- 6 degrees with the membrane normal. All of these values refer to the charge separated state, observed at low temperatures, where forward electron transfer to the iron-sulfur centers is partially blocked. Preliminary room temperature studies of P700(+)A1(-), employing X-band quantum beat oscillations, indicate a different orientation of A1(-) in its binding pocket. A comparison with crystallographic data provides information on the electron-transfer pathway in PSI. It appears that quantum beats represent excellent structural probes for the short-lived intermediates in the primary energy conversion steps of photosynthesis.

Cell Membrane↗
PubMed2001

Protein kinase C mu selectively activates the mitogen-activated protein kinase (MAPK) p42 pathway.

Here we show that human protein kinase C mu (PKC mu) activates the mitogen-activated protein kinase (MAPK). Transient expression of constitutive active PKC mu leads to an activation of Raf-1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKC mu enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKC mu is triggered by the Elk-1 ternary complex factor. PKC mu-mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKC mu does neither affect c-jun N-terminal kinase nor p38 MAPK.

Cells, Cultured↗
PubMed2001

ICL670A: a new synthetic oral chelator: evaluation in hypertransfused rats with selective radioiron probes of hepatocellular and reticuloendothelial iron stores and in iron-loaded rat heart cells in culture.

ICL670A (formerly CGP 72 670) or 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]- benzoic acid is a tridentate iron-selective synthetic chelator of the bis-hydroxyphenyl-triazole class of compounds. The present studies used selective radioiron probes of hepatocellular and reticuloendothelial (RE) iron stores in hypertransfused rats and iron-loaded heart cells to define the source of iron chelated in vivo by ICL670A and its mode of excretion, to examine its ability to remove iron directly from iron-loaded myocardial cells, and to examine its ability to interact with other chelators through a possible additive or synergistic effect. Results indicate that ICL670A given orally is 4 to 5 times more effective than parenteral deferoxamine (DFO) in promoting the excretion of chelatable iron from hepatocellular iron stores. The pattern of iron excretion produced by ICL670A is quite different from that of DFO and all iron excretion is restricted to the bile regardless of whether it is derived from RE or hepatocellular iron stores. Studies in heart cell cultures have shown a favorable interaction between DFO and ICL670A manifested in improved chelating efficiency of ICL670A, which is most probably explained by an exchange of chelated iron between ICL670A and DFO. These unique chelating properties of ICL670A may have practical implications for current efforts to design better therapeutic strategies for the management of transfusional iron overload.

Animals↗
PubMed2001

Exploring the "iron shuttle" hypothesis in chelation therapy: effects of combined deferoxamine and deferiprone treatment in hypertransfused rats with labeled iron stores and in iron-loaded rat heart cells in culture.

Although iron chelation therapy results in a significant improvement in well-being and life expectancy of thalassemic patients with transfusional iron overload, failure to achieve these goals in a substantial proportion of patients underlines the need for improved methods of treatment. In the present studies we used selective radioactive iron probes of hepatocellular and reticuloendothelial (RE) iron stores in hypertransfused rats and iron-loaded heart cells to compare the source of iron chelated in vivo by deferoxamine (DFO) or by deferiprone (L1) and its mode of excretion, to examine the ability of DFO and L1 to remove iron directly from iron-loaded myocardial cells, and to examine the mechanism of their combined interaction through a possible additive or synergistic effect. Our results indicate that L1 given orally is 1.6 to 1.9 times more effective in rats, on a weight-per-weight basis, than parenteral DFO in promoting the excretion of storage iron from parenchymal iron stores but shows no advantage over DFO in promoting RE iron excretion. Simultaneous administration of DFO and L1 results in an increase in chelating effect that is additive but not synergistic. The magnitude of this additive effect is identical to an increase in the equivalent (weight or molar) dose of DFO alone rather than the sum of the separate effects of L1 and DFO. This finding is most probably the result of a transfer of chelated iron from L1 to DFO. These observations may have practical implications for current efforts to design better therapeutic strategies for the management of transfusional iron overload.

Animals↗
PubMed2001

Chloroplast transcription at different light intensities. Glutathione-mediated phosphorylation of the major RNA polymerase involved in redox-regulated organellar gene expression.

Previous studies using purified RNA polymerase from mustard (Sinapis alba) chloroplasts showed control of transcription by an associated protein kinase. This kinase was found to respond to reversible thiol/disulfide formation mediated by glutathione (GSH), although at concentrations exceeding those thought to exist in vivo. In the present study, several lines of evidence are presented to substantiate the functioning of this regulation mechanism, also in vivo: (a) Studies on the polymerase-associated transcription kinase revealed that at appropriate ATP levels, GSH concentrations similar to those in vivo are sufficient to modulate the kinase activity; (b) GSH measurements from isolated mustard chloroplasts showed considerable differences in response to light intensity; (c) this was reflected by run-on transcription rates in isolated chloroplasts that were generally higher if organelles were prepared from seedlings incubated under high-light as compared with growth-light conditions; (d) the notion of a general transcriptional switch was strengthened by in vitro experiments showing that the kinase not only affects the transcription of a photosynthetic gene (psbA) but also that of a non-photosynthetic gene (trnQ); and (e) the polymerase-kinase complex revealed specific differences in the phosphorylation state of polypeptides depending on the light intensity to which the seedlings had been exposed prior to chloroplast isolation. Taken together, these data are consistent with GSH and phosphorylation-dependent regulation of chloroplast transcription in vivo.

Adenosine Triphosphate↗
PubMed2000

Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32.

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.

Animals↗
PubMed2000

ATP synthesis in lipoamide dehydrogenase deficiency.

Lipoamide dehydrogenase deficiency is an inborn error of several metabolic pathways, including pyruvate metabolism, Krebs cycle, and branched-chain amino acid degradation. The clinical course is variable, ranging from infantile neurodegenerative disease to recurrent episodes of liver failure or myoglobinuria starting later in life. In contrast, residual enzymatic activity in muscle tissue spans over a narrow range. Despite the recent elucidation of the underlying molecular pathology in most patients, relationships between the genotype and the biochemical and clinical phenotype remain unclear. In order to find a suitable assay for the prediction of clinical outcome and assessment of treatment, we have evaluated enzymatic activities and energetic states in fibroblasts from lipoamide dehydrogenase-deficient patients representing three different phenotypes and genotypes. Direct relationships between clinical parameters such as age of onset and disease severity and biochemical characteristics, including lipoamide dehydrogenase activity, pyruvate dehydrogenase complex activity, and ATP production ratio in fibroblasts, were identified. Clinical parameters were not reflected by lactate/pyruvate ratio. ATP production rate was in direct relationship with the severity of the neurological involvement; the patient with reduced ATP synthesis to 30% of the control mean had a severe neurodegenerative disease, whereas ATP synthesis values above 45% were associated with a more favorable course. Incubation of the patients' fibroblasts with dichloroacetate coupled with thiamin resulted in slight but significant improvement of the cell energetic state.

Adenosine Triphosphate↗
PubMed2000

Transcripts and sequence elements suggest differential promoter usage within the ycf3-psaAB gene cluster on mustard (Sinapis alba L.) chloroplast DNA.

The mustard chloroplast DNA region spanning the ycf3 gene and part of the psaAB operon was investigated. The ycf3 gene reveals two class-II introns that are removed during processing to give a mature 0.7-kb transcript, but no RNA editing seems to be involved. RNase protection and RT-PCR experiments suggest cotranscription of ycf3 with the downstream psaA gene, possibly from a NEP promoter upstream of ycf3, whereas distinct ycf3 and psaA transcripts are each initiated from PEP promoters. This situation is reminiscent of that for the trnK-psbA gene region. The implications for light-regulated versus light-independent expression of photosystem core-protein genes are discussed.

Amino Acid Sequence↗
PubMed2000

The multisubunit chloroplast RNA polymerase A from mustard (Sinapis alba L.). Integration of a prokaryotic core into a larger complex with organelle-specific functions.

We previously identified two multisubunit plastid RNA polymerases termed A and B. The B enzyme has a bacterial-type polypeptide composition and is sensitive to the prokaryotic transcription inhibitor rifampicin (Rif); the A enzyme has a more complex subunit structure and is Rif-resistant. Here we report results of N-terminal sequencing and MS carried out with the A enzyme, which establish that the latter contains rpo gene products and is structurally related to the B enzyme. Furthermore, evidence is provided that the A enzyme can be converted into a Rif-sensitive enzyme form in a phosphorylation-dependent manner in vitro by a treatment that results in depletion of a beta-like subunit. Database searches using sequence information derived from additional polypeptides that are present in purified A preparations revealed sequence similarity with chloroplast proteins involved in RNA processing and redox control. This proteomics approach thus points to the complexity of the chloroplast transcription apparatus and its interconnections with post-transcriptional and signalling mechanisms.

Alkaline Phosphatase↗
PubMed2000

[Induced abortions in the Third Reich. Legal basis and provision].

This article analyses, after introductory comments on the legal situation in the German Empire and the Weimar Republic, the legal basis for induced abortions during National Socialist rule in Germany. During this period the first legal definition for eugenically and medically indicated abortions was established. At the same time the prohibition of induced abortions outside these criteria was controlled more strictly and violations were punished more severely. This concerned abortions mainly for social reasons. The intention was to legalize abortion for those deemed "less worthy" while, at the same time, to minimise the number of abortions of those considered as "more valuable" to society. The main thrust of this policy was to increase the birth rate of "valuable" citizens. The second part of this paper focuses on eugenic and medical abortions at the University of Freiburg's Maternity Hospital.

Abortion, Induced↗
PubMed1999

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu.

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Protein kinase C mu is negatively regulated by 14-3-3 signal transduction proteins.

Recent studies have documented direct interaction between 14-3-3 proteins and key molecules in signal transduction pathways like Ras, Cbl, and protein kinases. In T cells, the 14-3-3tau isoform has been shown to associate with protein kinase C theta and to negatively regulate interleukin-2 secretion. Here we present data that 14-3-3tau interacts with protein kinase C mu (PKCmu), a subtype that differs from other PKC members in structure and activation mechanisms. Specific interaction of PKCmu and 14-3-3tau can be shown in the T cell line Jurkat by immunocoprecipitiation and by pulldown assays of either endogenous or overexpressed proteins using PKCmu-specific antibodies and GST-14-3-3 fusion proteins, respectively. Using PKCmu deletion mutants, the 14-3-3tau binding region is mapped within the regulatory C1 domain. Binding of 14-3-3tau to PKCmu is significantly enhanced upon phorbol ester stimulation of PKCmu kinase activity in Jurkat cells and occurs via a Cbl-like serine containing consensus motif. However, 14-3-3tau is not a substrate of PKCmu. In contrast 14-3-3tau strongly down-regulates PKCmu kinase activity in vitro. Moreover, overexpression of 14-3-3tau significantly reduced phorbol ester induced activation of PKCmu kinase activity in intact cells. We therefore conclude that 14-3-3tau is a negative regulator of PKCmu in T cells.

14-3-3 Proteins↗
PubMed1999

Cardioprotective effect of alpha-tocopherol, ascorbate, deferoxamine, and deferiprone: mitochondrial function in cultured, iron-loaded heart cells.

Because mitochondrial inner membrane respiratory complexes are important targets of iron toxicity, we used iron-loaded rat heart cells in culture to study the beneficial effect on mitochondrial enzymes of the iron chelators deferoxamine (DFO) and deferiprone (L1) and of antioxidants and reducing agents (ascorbate and alpha-tocopherol). Reduced nicotinamide adenine dinucleotide-cytochrome c oxidoreductase (complex I-III) and succinate dehydrogenase were the most-sensitive indicators of iron toxicity and cardioprotective effect. Although at concentrations below 0.3 mmol/L the iron-mobilizing effect of L1 was less than that of DFO, both were equally effective in protecting or restoring mitochondrial respiratory enzyme activity. At 1.0 mmol/L, L1 toxicity was manifested in respiratory enzyme inhibition, whereas DFO had no such effect. Ascorbate (0.057 to 5.7 mmol/L) had a mild cardioprotective effect at the highest concentration only, in association with decreased cellular iron uptake. By contrast, alpha-tocopherol (0.023 mmol/L) completely inhibited mitochondrial iron toxicity without affecting iron uptake or release, and irrespective of whether it was used before, during, or after in vitro iron loading. These observations illustrate the usefulness and limitations of iron chelators and other agents used for preventing iron toxicity to the heart and other vital organs, and they underline the need for exploring in more detail the effects of these agents in the clinical setting.

Animals↗
PubMed1999

PTK, the chloroplast RNA polymerase-associated protein kinase from mustard (Sinapis alba), mediates redox control of plastid in vitro transcription.

The major RNA polymerase from mustard chloroplasts is a multi-subunit enzyme consisting of core components and associated factors. Among the latter is a heterotrimeric factor named PTK (plastid transcription kinase) because of its serine/threonine-type protein kinase activity. PTK activity itself depends on its phosphorylation state. In addition, we show that it responds to glutathione but not to other redox-reactive reagents that were tested, and both glutathione and phosphorylation act antagonistically. Using a homologous in vitro system, we find that PTK selectively phosphorylates subunit(s) of plastid RNA polymerase and is involved in determining the level of faithful transcription from the chloroplast psbA promoter. Together, these results establish a role for phosphorylation and redox state in the regulation of plastid gene expression.

Chloroplasts↗
PubMed1999

Direct transcriptional control of the chloroplast genes psbA and psaAB adjusts photosynthesis to light energy distribution in plants.

Two photosystems, I and II, absorb and convert light energy in photosynthesis in chloroplasts of green plants. The genes psbA and psaAB of the cytoplasmic chloroplast genome encode core components of photosystem II and photosystem I, respectively. Here we show that the absolute amounts of photosystem I and photosystem II respond, in a complementary manner, to changes in light quality that preferentially excite each photosystem in mustard seedlings. We also show that the initial response to altered energy distribution is a change in the rates of transcription of psbA and psaAB. Changes in chlorophyll fluorescence emission in vivo suggest that the signal initiating this change is the oxidation-reduction state of plastoquinone, a component of the photosynthetic electron transport chain that connects photosystem I and photosystem II. The results are consistent with transcriptional effects observed previously with chloroplasts isolated in vitro and demonstrate that redox control of chloroplast transcription initiates long-term adjustments that compensate for imbalance in energy distribution and adapt the whole plant to altered light environments.

Chloroplasts↗