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Biomedical subjects

C Gerth

Publications and source records attributed to C Gerth.

At least 19 recordsLinked to original sources
PubMed2004

Impact of intravenous oxygen therapy on the expression of reticulocyte-type 15-lipoxygenase in human volunteers.

Intravenous oxygen infusion is used in complementary medicine to fight inflammatory disorders and repeated application of this therapeutic method leads to an increase in eosinophilic granulocytes in the peripheral blood. Since this subset of human peripheral leukocytes are known to express large amounts of the reticulocyte-type 15-lipoxygenase (15-lipoxygenase 1), which was suggested to exhibit anti-inflammatory activities, we profiled expression of this enzyme in the peripheral blood during the time course of typical oxygen infusion therapy. For this purpose seven volunteers were treated with intravenous infusion of oxygen gas for 4 weeks and the time-course of 15-lipoxygenase expression as well as the eosinophil count were monitored during and after the treatment interval. We found that 15-lipoxygenase 1 expression and the eosinophil count were significantly increased during the treatment period but returned to normal after the therapy was stopped. There was a striking correlation between the relative number of 15-lipoxygenase transcripts and the eosinophil counts suggesting eosinophils as major source of 15-lipoxygenase 1 expression. Since 15-lipoxygenase has been implicated in the resolving phase of acute inflammatory diseases the anti-inflammatory effects of intravenous oxygen infusion may be explained at least in part by our experimental findings.

Adult↗
PubMed2002

Multiple ionization of atom clusters by intense soft X-rays from a free-electron laser.

Intense radiation from lasers has opened up many new areas of research in physics and chemistry, and has revolutionized optical technology. So far, most work in the field of nonlinear processes has been restricted to infrared, visible and ultraviolet light, although progress in the development of X-ray lasers has been made recently. With the advent of a free-electron laser in the soft-X-ray regime below 100 nm wavelength, a new light source is now available for experiments with intense, short-wavelength radiation that could be used to obtain deeper insights into the structure of matter. Other free-electron sources with even shorter wavelengths are planned for the future. Here we present initial results from a study of the interaction of soft X-ray radiation, generated by a free-electron laser, with Xe atoms and clusters. We find that, whereas Xe atoms become only singly ionized by the absorption of single photons, absorption in clusters is strongly enhanced. On average, each atom in large clusters absorbs up to 400 eV, corresponding to 30 photons. We suggest that the clusters are heated up and electrons are emitted after acquiring sufficient energy. The clusters finally disintegrate completely by Coulomb explosion.

Journal Article↗
PubMed2002

Transient experimental anemia in cholesterol-fed rabbits induces systemic overexpression of the reticulocyte-type 15-lipoxygenase and protects from aortic lipid deposition.

Oxidative modification of low-density lipoprotein has been implicated in atherogenesis and the lipid peroxidizing enzyme 12/15-lipoxygenase (12/15-LOX) was suggested to be involved. For this study, we induced a strong and long-lasting systemic overexpression of the 15-LOX, in female New Zealand White rabbits by transient experimental anemia. After the hematopoietic parameters had returned to normal, these animals and age-matched controls were fed a lipid-rich Western-type diet for 10 weeks. Analyzing the lipid deposition in the aortic wall, we found that the 15-LOX overexpressing rabbits deposited significantly (P<0.01) less cholesteryl linoleate in the thoracic aorta than the corresponding controls. Similar results were obtained when free cholesterol and cholesteryl oleate were quantified. However, in the aortic arch where lipid deposition was much more severe a similar trend was observed, but the effects were not significant any more. Comparative determination (lipoxygenase overexpressing vs. control animals) of various plasma parameters as well as histological inspections of major organs did not reveal any indications for major organ malfunction. These data suggest that transient experimental anemia, which is accompanied by a long-lasting overexpression of the reticulocyte-type 15-LOX protects cholesterol-fed rabbits from lipid deposition in the aortic wall.

Anemia↗
PubMed2001

Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis.

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.

Amino Acid Sequence↗
PubMed2000

Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity.

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.

Animals↗
PubMed2000

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Animals↗
PubMed2000

Impact of notch filter use on waveforms of First- and Second-Order-Kernel responses from multifocal ERGs.

The filter settings of the amplifier section of ERG recording systems have large impact on the waveforms of the recorded responses. In this study, the effects of a 50 Hz notch filter were assessed for both First-Order-Kernel- (FOK) and Second-Order-Kernel-responses (SOK 1st slice) from multifocal ERGs recorded with a VERIS III system. Amplitude and phase responses of the amplifier section were recorded for typical filter settings with the notch filter active and inactive. Multifocal ERGs (MERGs) from a group of 11 normal subjects were recorded for both amplifier settings, the waveforms resulted from those recordings were compared in the time and frequency domain. To verify the results, the recordings without the notch filter were digitally filtered with a simulated notch filter and compared to the responses recorded with an active analog notch filter. The line filter has the biggest attenuation at 50 Hz with an additional phase jump of 180 degrees. The FOK responses of MERGs are assembled by frequencies below 65 Hz with main spectral components between 19 and 47 Hz. The 1st slice of the SOK consists of frequencies up to 100 Hz with main components between 19 and 84 Hz. Thus, if FOK recordings are to be analyzed, the notch filter of the amplifier can be used in order to cope with noise problems caused by the line frequency of 50 Hz. However, one must be aware that the attenuation of higher frequency components will lead to changes of the waveforms. For SOK analysis, main spectral components are attenuated and/or shifted in phase, which leads to completely different waveforms and severe distortion of the recording results.

Adult↗
PubMed2000

Early-onset severe rod-cone dystrophy in young children with RPE65 mutations.

PURPOSE: To describe the ocular phenotype of patients with RPE65 mutations in infancy and young childhood. METHODS: Four children from three families with severe early-onset visual impairment related to electrophysiologically detectable retinal dystrophy were screened for mutations in the RPE65 gene. Visual function from infancy to the age of 10 years was assessed with age-adapted methods. Clinical examinations and electroretinograms (ERGs) were also performed on the six parents. RESULTS: In all three families, patients were compound heterozygous for mutations of the RPE65 gene (ins144T/IVS1+5G-->A, R91W/Y368H, 1114delA+T457N/IVS1+5G-->A). Visual acuity was measurable in all patients at the age of 6 to 10 years, despite severe visual impairment noted during infancy and congenital nystagmus in three of the four patients. Photophobia was not a feature. Funduscopic changes were discrete, the most prominent finding being increased granularity in the macula and the periphery. Peripheral vision was well preserved, measured by Goldmann perimetry. Rod ERGs were not recordable, whereas cone ERGs were detectable in early childhood. All features taken together suggest a specific form of Leber congenital amaurosis (LCA) distinguishable on clinical grounds. ERGs were normal in five of the six parents. One father had an ERG compatible with congenital stationary night blindness unrelated to his heterozygous state for the RPE65 mutation. CONCLUSIONS: RPE65 mutations on both alleles may be associated with early-onset severe rod-cone dystrophy. Visual functions of the four patients were better than is usually seen in LCA, in particular in cases associated with retGC1 mutations. RPE65 mutations should be suspected in infants who appear to be blind in dim surroundings but react to objects in bright illumination and have nonrecordable rod ERGs and residual cone ERGs.

Age of Onset↗
PubMed1999

Shape and specificity in mammalian 15-lipoxygenase active site. The functional interplay of sequence determinants for the reaction specificity.

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.

Animals↗
PubMed1997

Molecular analysis of 29 pyruvate kinase-deficient patients from central Europe with hereditary hemolytic anemia.

We investigated the DNA of 29 unrelated pyruvate kinase (PK) deficiency (PKD) patients from Central Europe with hereditary nonspherocytic hemolytic anemia for mutations in the PK-L/R gene. Among 58 potentially affected alleles, 53 mutations were identified, of which 17 were different from each other. Of these 17 mutations, 13 were single-nucleotide (nt) substitutions resulting in amino acid exchanges, G787A (Gly263-Arg), G994A (Gly332-Ser), G1006T (Ala336-Ser), G1010A (Arg337-Gln), A1081G (Asn361-Asp), G1127T (Ser376-Ile), G1174A (Ala392-Thr), G1281T (Glu427-Asp), C1454T (Ser485-Phe), C1456T (Arg486-Trp), G1493A (Arg498-His), G1529A (Arg510-Gin), and C1594T (Arg532-Trp); 1 in-frame triplet deletion, 1060delAAG (delLys354); 1 in-frame triplet insertion, 1203insAGC (insSer after Cys401); 1 splicesite mutation, 101-1G-A; and 1 frameshift deletion, 628delGT. Six mutations, 628delGT, G787A, G1010A, G1127T, G1281T, and C1454T, are described for the first time. To test the hypothesis of a single origin of the most common PK mutation in the European population, G1529A, we investigated all patients at four polymorphic sites in the PK-L/R gene: C/A at nt 1705, C/T at nt 1992, the (ATT)n microsatellite in intron J, and a polymorphism (T)10/(T)19 in intron I. Nine patients homozygous for mutation G1529A were consistent in all four markers. In the group of patients homozygous for mutation G1529A, the hematologic parameters and clinical manifestations have been studied in detail. Although having an identical mutation in the PK-L/R gene, the patients are affected differently. Their appearance ranges from a very mild compensated hemolysis to a severe anemia. Possible molecular explanations are discussed.

Anemia, Hemolytic↗
PubMed1993

Proliferating alveolar macrophages in BAL and lung function changes in interstitial lung disease.

In interstitial lung disease, the number of alveolar macrophages (AMs) can be increased. This may be caused by recruitment of precursor cells from peripheral blood and/or local proliferation in the lung. We therefore analysed proliferation, by studying both the expression of the nuclear proliferation antigen, Ki67, and the deoxyribonucleic acid (DNA) content, using the Feulgen reaction followed by cytometry. The patients had interstitial lung disease, i.e. sarcoidosis (n = 20), extrinsic allergic alveolitis (n = 20), idiopathic lung fibrosis or lung involvement in collagen-vascular disease (n = 19). In all patient groups there was a significant increase in proliferating AMs compared to healthy controls (4.2 versus 1.4% Feulgen, 2.1 versus 0.5% Ki67), with a significant correlation between these two parameters. A positive correlation was also found in bronchoalveolar lavage (BAL) between numbers of lymphocytes and proliferating cells in sarcoidosis and in fibrosis. In fibrosis, numbers of eosinophils and proliferating cells were also positively correlated. Our main finding was, however, a positive correlation between numbers of proliferating cells (Feulgen) and lung function parameters, especially vital capacity and oxygen tension (PO2) at rest, in patients with sarcoidosis and lung fibrosis. By contrast, in extrinsic allergic alveolitis, no correlation could be observed between proliferating cells and cell population or lung function. Our results suggest that local proliferation of macrophages is an important element in interstitial lung disease.

Adult↗
PubMed1991

[A titrographic method for determining the glycolysis flow rate with Plasmodium berghei-infected red blood cells].

A procedure is described for the elucidation of the glycolytic flux rate of red blood cells infected with the malarial parasite Plasmodium berghei. It is based on the titration of the protons originating from the glycolytic lactate accumulation. Compared with traditional methods of biochemical measurements of glucose consumption or accumulation of lactate the proposed procedure shows the following advantages: continuously measurement is possible; constancy of the pH-value during the measurement; lower amounts of biological material are necessary (increased sensitivity) increased accuracy; The method can also be applied to studies on other cells and species with normal or increased glycolytic flux rate.

Animals↗
PubMed1991

[Diagnosis of genetically determined diseases].

Enzymopathies of pyruvate kinase are caused by defects of structural genes forming stable and unstable mutants, respectively. Stable mutants of PK are characterized by high S0,5 PEP and nearly unchanged Vmax. A decrease of PEP affinity can be the result from a very high allosteric constant L0 or from an increased KPEP. From the pattern of PAGE can be concluded that stable PK mutants are tetraheteromers composed of two normal and two shortened polypeptide chains. We suppose that this is the result of a mutation of a codon which stops the polypeptide synthesis of PK earlier. Most PK mutants are unstable. They are characterized by low catalytic activity and high PEP affinity. The kinetic properties of unstable mutants are changed posttranslational by proteolytic modifications. Furthermore low S0,5 PEP values result from a persistence of the isoenzyme PK-K in reticulocytes and erythrocytes, respectively. A prenatal diagnosis of PK enzymopathies can be carried out with a very small blood volume by using the method of isoelectrophoretic focussing.

Chromatography, Affinity↗
PubMed1990

Phosphofructokinase from Plasmodium berghei. Influence of Mg2+, ATP and Mg2(+)-complexed ATP.

The control enzyme phosphofructokinase is of regulatory significance in the metabolism of glucose by the malarial parasite Plasmodium berghei. (1) The enzyme was partially purified from erythrocytic stages of P. berghei by precipitation with poly(ethylene glycol) and chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. (2) Similarly to various other phosphofructokinases, the enzyme from P. berghei shows an allosteric behaviour. It is activated by fructose 6-phosphate and inhibited by ATP. (3) The effects of Mg2(+)-complexed ATP, free ATP and Mg2+ were studied by keeping constant the concentration of one of these and varying the concentrations of the other two. (4) The enzyme is shown to be allosterically inhibited by free ATP and by higher concentrations of Mg2+. Compared with phosphofructokinase of erythrocytes, inhibition by ATP is weaker by two orders of magnitude. Mg2(+)-complexed ATP has no effect on allosteric regulation. (5) The proposed kinetic model provides an adequate description of the data.

Adenosine Triphosphate↗
PubMed1990

Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization.

Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase (G6PD) in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. From infected rat erythrocytes the parasites were isolated, washed, and lysed. G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B, although the separation of the malaria-specific enzyme from that of the host cell was not complete. Malarial G6PD significantly differed from the red cell enzyme with respect to its electrophoretic properties. In cellulose acetate electrophoresis, a band with catodic mobility was observed in addition to the anodically mobile host cell enzyme at pH 7.0. The subunits of the parasite-specific G6PD have a molecular weight of 55 kDa in contrast to 59 kDa of red cell G6PD subunits. The enzyme from P. berghei shows no cross-reactivity with polyclonal antibodies against G6PD from rat erythrocytes. Thus, a close evolutionary relationship between both proteins and the presence of proteolytic modifications could be excluded. The Km value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.

Animals↗