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PubMedDate not supplied

Evaluation of the antioxidant capacity of ubiquinol and dihydrolipoic acid.

Ubiquinone and alpha-lipoic acid are natural constituents which are involved in mitochondrial energy metabolism. Their bioenergetic activities require redox-cycling. In the case of alpha-lipoic acid redox-cycling leads to dihydrolipoic acid which occurs in multienzyme complexes involved in the citric acid cycle while UQ recycles through semi- and divalently reduced ubiquinones in the respiratory chain. We have proved the validity of the concept about the antioxidant function of these natural compounds in their reduced form. Ubiquinol was found to interfere with lipid peroxidation of liposomal membranes being itself degradated by two consecutive oxidation steps. Dihydrolipoic acid was found to totally recycle ubiquinone to the antioxidant active divalently reduced form. In contrast to the antioxidative derived reaction products of ubiquinols which in turn promoted lipid peroxidation, the antioxidant derived reaction product of dihydrolipoic acid was the unreactive two electron oxidation product alpha-lipoic acid. Our experiments demonstrate the existence of an dihydrolipoic acid driven recycling of UQ to the antioxidative-active UQH2. The efficiency of the antioxidative capacity of the latter was found to be diminished through prooxidant activities of the antioxidant-derived metabolites.

Antioxidants↗
PubMed1999

Cloning of an ORF with homology to Mycobacterium echA1, encoding the enoyl-CoA hydratase, in Rhodococcus fascians.

An open reading frame encoding a polypeptide of significant homology (55.7% identity) with the enoyl-CoA hydratase encoded by the gene echA1 from Mycobacterium tuberculosis has been found in the genome of the plant-pathogen bacteria Rhodococcus fascians strain NRRL-B-15096. Sequence alignments showed that it possesses several conserved blocks common to E. coli, M. tuberculosis and human mitochondria. One of such blocks includes a glutamate residue located at position 149, corresponding to the glutamate 139 of Escherichia coli. This glutamate was previously shown to be the catalytic residue of enoyl-CoA hydratase in the multienzyme complex of fatty acid oxidation from E. coli. Our results provide additional information on the conserved domains of this enzyme. Significant homologies in other genome regions between R. fascians and M. tuberculosis confirm their phylogenetic relationship.

Amino Acid Sequence↗
PubMed1997

Inherited disorders of glycoprotein synthesis: cell biological insights.

Disorders of glycoprotein synthesis have been described only recently, and few have been studied extensively at both the clinical and biochemical level. The identification and characterization of these rare diseases are important, not only for the patients and their families, but because they offer enormous insight into biological processes. For example, the targeting of acid hydrolases to lysosomes by mannose-6-phosphate was discovered as a direct result of the elucidation of the defect in I-cell disease. The notion of carbohydrates as targeting agents continues to have ramifications today, with the success of macrophage-targeted enzyme replacement therapy for Gaucher disease. Likewise, confirmation of the in vivo role of fucose-containing glycans and selectins in neutrophil function came from studies using specimens from patients with leucocyte adhesion deficiency type II due to reduced availability of GDP-fucose. Identification of the in vivo ligands of selectins also has implications for anti-inflammatory therapies. Macular corneal dystrophy and spondyloepiphyseal dysplasia tarda offer an opportunity to investigate the number of different sulfotransferases in cells, their substrates, and their tissue expression. The Ehlers-Danlos progeroid variant offers insight into the function and regulation of the proteoglycan decorin, and suggests that several of the enzymes involved in proteoglycan synthesis may function as a multienzyme complex. The common occurrence of hypergonadotropic hypogonadism in patients with galactosemia or carbohydrate-deficient glycoprotein protein syndrome, due to defective N-linked glycosylation, suggests that ovarian function is particularly dependent on proper glycan-synthesis. A host of other concepts await discovery as a fuller contingent of human disorders of glycan synthesis achieves recognition.

Anemia, Dyserythropoietic, Congenital↗
PubMed2000

The expression of MHC class I, TAP1/2, and LMP2/7 gene in human gastric cancer cell lines.

Intracellular antigens are presented to CD8+ T cells through major histocompatibility complex (MHC) class I molecule. For stable MHC class I expression, several molecules as well as the MHC molecule itself have to express simultaneously and function well. To determine a gene associated with MHC class I surface expression, the expressions of LMP2/7 and TAP1/2 including MHC I gene were analyzed in ten human gastric cancer cell lines, using RT-PCR and Northern blot analysis. Although LMP2, TAP1/2, and MHC class I gene expression were reduced in some cells, this was not significantly associated with MHC class I surface expression. By comparison, the expression of LMP7 was significantly reduced in three of ten cell lines, which also showed low levels of MHC class I surface expression, and increased in four of ten cell lines, which also showed high levels of MHC class I surface expression. These results suggest that the level of MHC class I surface expression is associated, in most cases, with the expression of the LMP7 gene regardless of the LMP2, TAP1, TAP2, or MHC class I genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Identification and characterization of a beta proteasome subunit cluster in the Japanese pufferfish (Fugu rubripes).

The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for beta-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-gamma and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-gamma-regulated beta-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.

Animals↗
PubMed1999

Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase.

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

Amino Acid Sequence↗
PubMed1999

Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides.

Most of the MHC class I peptides presented to the immune system are generated during the course of protein breakdown by the proteasome. However, the precise role of the proteasome, e.g., whether this particle or some other protease generates the carboxyl (C) and amino (N) termini of the presented 8- to 10-residue peptides, is not clear. Here, we show that presentation on Db of ASNENMETM, a peptide from influenza nucleoprotein, and on Kb of FAPGNYPAL, a peptide from Sendai virus nucleoprotein, was blocked by the proteasome inhibitor, lactacystin. Using plasmid minigene constructs encoding oligopeptides of various lengths, we found that presentation of ASNENMETM from C-terminally extended peptides that contain this antigenic peptide plus three or five additional amino acids and presentation of FAPGNYPAL from a peptide containing FAPGNYPAL plus one additional C-terminal residue required the proteasome. In contrast, the proteasome inhibitor did not reduce presentation of cytosolically expressed ASNENMETM or FAPGNYPAL or N-terminally extended versions of these peptides, suggesting involvement of aminopeptidase(s) in trimming these N-extended variants. Accordingly, when the N termini of these 3N-extended peptides were blocked by acetylation, they were resistant to hydrolysis by cellular aminopeptidases and pure leucine aminopeptidase. Moreover, if introduced into the cytosol, Ag presentation of these peptides occurred to a much lesser extent than from their nonacetylated counterparts. Thus, the proteasome is essential for the generation of ASNENMETM and FAPGNYPAL peptides from the full-length nucleoproteins. Although it generates the C termini of these presented peptides, distinct aminopeptidase(s) can trim the N termini of these presented peptides to their proper size.

Aminopeptidases↗
PubMed2000

Plant cell biology in the new millennium: new tools and new insights.

The highly regulated structural components of the plant cell form the basis of its function. It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary metabolism to the "transducon" complexes of signal transduction elements that allow for the highly efficient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of the plant cell. First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell biologists to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell biologist now has available a vast array of fluorescent probes that will report cell parameters as diverse as the pH of the cytosol, the oxygen level in a tissue, or the dynamics of the cytoskeleton. The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.

Journal Article↗
PubMedDate not supplied

[Biosynthes of polyketide antibiotics by various actinomycin producing Streptomyces species].

A collection of actinomycin-producing Streptomyces strains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned act gene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolor revealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between biosynthetic pathways of actinomycins and polyketides is discussed.

Acyltransferases↗
PubMed1976

Biohydrogenation of unsaturated fatty acids. Hydrogenation by cell-free preparations of Butyrivibrio fibrisolvens.

Hydrogenation of cis-9,trans-11-octadecadienoic acid to yield trans-11-octadecenoic acid by cell-free preparations of Butyrivibrio fibrisolvens has been obtained under strictly anaerobic conditions. Reduced methyl viologen, NADH, and an endogenous electron donor each can serve as a reductant. Inhibition studies and gel filtration patterns reveal the presence of at least two hydrogenation systems, one of which is coupled through a flavin, possibly FMN. Although the enzymes comprising the biohydrogenation pathway, the fatty acid reductases and linoleic acid isomerase, are part of the bacterial membrane, they do not appear to be constituted as a multienzyme complex.

Cell-Free System↗
PubMed1990

Immunoaffinity chromatography of enzymes.

Immunoaffinity chromatography of enzymes represents an attractive purification technique suitable for one-step and large-scale purification of enzymes to homogeneity. Monoclonal and polyclonal antibodies can be used equally well. The broad use of the technique is restricted by the harsh elution conditions which are often required. The efforts to overcome these limitations and to optimize the method are reviewed, viz. proenzyme purification, purification of enzymes as part of multienzyme complexes carried out by a mild dissociation step, specific elution by substrates and effectors, enzyme stabilization, electrophoretical desorption and negative elution by adsorbing impurities from the crude extract, and hypotonic elution. The current practice is discussed considering antibody and enzyme selection, optimization of elution conditions, and washing steps using different media. Representative examples are given for various approaches.

Animals↗
PubMed1992

[The role of disorders in the structural-functional properties of the erythrocyte membranes and energy metabolism in the progression of anemia in patients with terminal kidney failure].

As many as 30 healthy persons and 65 patients suffering from chronic glomerulonephritis with terminal renal failure (TRF) placed on program dialysis were examined for lipid composition of the membranes and functional properties of red blood cells. By the gravity of anemia the patients were distributed into 2 groups. It has been established that in the red blood cell membranes, there was an increase of the content of cholesterol and sphingomyelin, and a decline of the content of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. In addition to the changes in the lipid content of red blood cells, the peripheral red blood cell pool showed an increase of the amount of pathologically shaped cells, a reduction of the cell capacity to deformation, a rise of the content of fibrinogen in the supramembranous layer. Destruction of the phospholipid matrix of the red blood cell membrane was attended by noticeable restructure of the work of the multienzymic complex of glycolysis, resulting in energy metabolism destabilization. Analysis of the data obtained has demonstrated that as the structural and functional properties of the membranes underwent alterations and the mechanisms of stabilization of red blood cell energy homeostasis got disintegrated, anemia in patients with TRF became graver.

Adolescent↗
PubMed1992

Immune disorders of the liver and bile duct.

Several hepatobiliary diseases appear to be mediated by the host immune response. They can be subdivided into those in which the immune reaction is against an infectious agent such as hepatitis viruses, those in which the immune reaction appears to be against an autoantigen expressed on hepatobiliary cells, and those due to alloimmunity. The existence of autoimmune liver diseases indicates a breakdown in the mechanisms responsible for self-tolerance. In HBV infection, the hepatocellular necrosis appears to be mediated by the host immune response against viral antigens expressed on the membranes of infected hepatocytes. Autoimmune chronic active hepatitis can be subdivided on the basis of differences in circulating autoantibodies. In classic type I autoimmune chronic hepatitis, autoantibodies are directed against non-organ- and non-species-specific antigens. Thus, they are unlikely to be involved in pathogenesis. In contrast, type II autoimmune chronic hepatitis is characterized by antibodies against specific cytochrome P-450 isoenzymes that appear to be expressed on the surface membrane of hepatocytes. Immunogenic cytochrome P-450 also can be induced by drug metabolism and haptenation. This indicates that environmental or medicinal xenobiotics may initiate autoimmune liver damage. Primary biliary cirrhosis is characterized by T-cell-mediated inflammation and destruction of interlobular and septal bile ducts and antibodies specific for epitopes of the 2-oxo-acid dehydrogenase multienzyme complex of mitochondria. The histopathologic lesion NSDC also is observed in alloimmune-mediated diseases such as CGVHD and rejection of liver allografts. PSC may be mediated by an immune response against endothelial cells of the peribiliary capillary plexus, with secondary reactions to bile duct epithelial cell antigens. The pathogenesis of alcoholic liver disease is multifactorial, but one component involves an immune response to acetaldehyde-protein adducts. Secondary sensitization of cell-mediated effector mechanisms, endothelial damage, and secretion of noxious cytokines appear to be involved in pathogenesis.

Autoimmune Diseases↗
PubMed1996

[A comparative analysis of biological properties of glucosylrtanterases secreted by Streptococcus sanguis and Streptococcus mutans]

Our study make a comparative analysis of biological properties of glucosyltranterases(GTF) secreted by S.sanguis 34 and S.mutans Ingbritt(c).The results as follows:(1)S.sanguis 34 has more extractions of extracelluar GTF than S.mutans Ingbritt(c) under equal cultured conditions,but S.mutans Inggbritt(c) has more activity of extracelluar GTF than S.sanguis.These differences are identical with the difference of their cariogenicities;(2)GTF of S.sanguis 34 can also synthesize water-soluble glucan,and is a multienzyme complex system as S.mutans. S.sanguis might catalyse sufficient dextran formation to stimulate the primer-dependent activity of the S.mutans GTF enzyme. This might aid the adhesion of this comparatively late colonizer;(3)easier to obstruct high pure GTF of S.sangusi 34 S.mutans.

Journal Article↗
PubMed1992

[Lignin and ligninase].

Ligninases (lignin peroxidases) are heme-containing peroxidases excreted by some white-rot fungi as components of their lignolytic multienzyme complexes. These peroxidases functioning at rather acidic media catalyze oxidative cleavage of both synthetic non-phenolic lignin models and many other oxidation-proof compounds (chloroorganic pesticides, carcinogenic hydrocarbons, etc.). Data on the ligninase structure and functions not only shed light of the lignin biodegradation but also open new perspectives in peroxidation chemistry and biotechnology. Many aspects of ligninase catalytic mechanism can be understood in comparative studies of congruent chemical reactions, e.g., peroxidisulfate-supported oxidation, as well as of ligninase-like activity of some plant and animal peroxidases which is also manifested at low pH. Ligninases are not only more powerful oxidative agents than other peroxidases, but also, in contrast to latters, appear to be able to control the contributions of C-C and C-O bond splitting in primary radical-cations of substrates. The contribution of the oxidative-hydrolytic dealkylation of radical cations can be considered as one of classification criteria for lignolytic enzymes.

Amino Acid Sequence↗
PubMedDate not supplied

Cholesterol-lowering effect of organosulphur compounds from garlic: a possible mechanism of action.

OBJECTIVES: Hyperlipidemia constitutes a major etiopathological factor for atherosclerosis. The medicinal value of garlic is best known for its lipid lowering effects and antiatherogenic effects. The mechanism by which lipid soluble organosulphur compounds from garlic reduce plasma lipids has not been fully investigated. The author had previously shown that the hepatic activity of beta-hydroxy-beta-methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis and the incorporation of radiolabeled (1, 2 14C), acetate into hepatic free and esterified cholesterol was significantly decreased in rat treated with garlic derived organosulphur compounds. We hypothesised that the antiatherogenic effect of the organosulphur compounds may be attributed to the formation of protein internal disulphide and thus inactivation of thiol (-SH) group enzymes such as HMG-CoA reductase and the multienzyme complex of fatty acid synthesis. The objective of the present study is to elucidate the inhibitory mechanism by in vitro studies. METHOD: Lipid soluble organosulphur compounds from garlic were treated in vitro with Luke's cysteine reagent (representing the thiol (-SH) group of enzymes) and the interaction products were separated by paper chromatography. RESULT: The result indicated that the organosulphur compounds were capable of interacting with the thiol (-SH) group of cysteine and thus forming cysteine derivatives. CONCLUSION: The antiatherogenic effects of these organosulphur compounds can be attributed to such reactions that inhibit HMG-CoA reductase and other lipogenic enzymes. The anticarcinogenic effects of these compounds may also be due to inhibitory reactions on enzymes that activate carcinogens.

Cholesterol↗
PubMed1991

[Regulation of cooperative properties of alpha-ketoglutarate dehydrogenase by means of thiol-disulfide metabolism].

The redox state of two SH-groups per enzyme subunit has been shown to control the cooperative properties of alpha-ketoglutarate dehydrogenase. These thiols oxidized, alpha-ketoglutarate dehydrogenase does not exhibit any cooperative properties. The enzyme reduction leads to subunit interactions. It has been found that the most effective agent reducing the alpha-ketoglutarate dehydrogenase thiols essential for the cooperativity is dihydrolipoate, one of the intermediates of the overall alpha-ketoglutarate dehydrogenase reaction. The possibility of changing the properties of alpha-ketoglutarate dehydrogenase in the multienzyme complex under the conditions when the lipoic acid integrated into the complex is reduced, has been investigated. Thus, incubation of the alpha-ketoglutarate dehydrogenase complex with NADH has been found to induce the conversion from the non-cooperative form to the cooperative one, presumably through the reduction of lipoic acid bound to the complex in the reaction catalyzed by lipoyl dehydrogenase, the third component of the complex.

Animals↗