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1.
J Inherit Metab Dis ; 36(6): 1073-7, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23371451

ABSTRACT

BACKGROUND: Cystinosis is an autosomal recessive disease caused by intralysosomal cystine accumulation, treated with cysteamine. Recently, new adverse effects of cysteamine were reported. Skin biopsies showed microvascular proliferation (angioendotheliomatosis). To examine the mechanism of angioendotheliomatosis associated with cysteamine toxicity, we examined the effect of cysteamine on human dermal microvascular endothelial cells (HDMVEC). METHODS: After cysteamine exposure (range 0-3.0 mM) during 24 h, cell viability was measured using water soluble tetrazolium salt-1 (WST-1) in both control HDMVEC and fibroblasts. Cell proliferation and apoptosis rate were measured in HDMVEC by bromodeoxyuridine (BrdU) incorporation and caspase 3 and caspase 7 activity, respectively. Intracellular glutathione (GSH) was measured in HDMVEC after cysteamine exposure of 0, 0.1 or 1.0 mM. Medium and cysteamine were refreshed every 6 h to mimic the in vivo situation. Next, cell viability in HDMVEC was measured after 24 h of GSH exposure (range 0-10.0 mM). RESULTS: HDMVEC viability and proliferation increased after cysteamine exposure 0.03-3.0 mM (p < 0.01) and 0.03-1.0 mM (p = 0.01) respectively; cell viability in fibroblasts was not affected by incubation with cysteamine. Apoptosis remained unaffected by incubation with 0-1.0 mM cysteamine, 3.0 mM caused increased apoptosis. Intracellular GSH was significantly increased after incubation with cysteamine 0.1 mM (p = 0.02) and 1.0 mM (p < 0.01). HDMVEC viability increased after exposure to GSH 1.0-5.0 mM (p < 0.01). CONCLUSION: Cysteamine concentrations, similar to those described in plasma of cystinosis patients, stimulate HDMVEC viability and proliferation and increase intracellular GSH content. We postulate that this mechanism might underlie angioendotheliomatosis induced by cysteamine.


Subject(s)
Cysteamine/pharmacology , Dermis/cytology , Endothelial Cells/drug effects , Endothelial Cells/physiology , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dermis/blood supply , Dermis/metabolism , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Glutathione/analysis , Glutathione/metabolism , Glutathione/pharmacology , Humans
2.
J Med Genet ; 45(8): 525-34, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18456717

ABSTRACT

BACKGROUND: The m.3243A>G mutation in the mitochondrial tRNA(Leu(UUR)) gene is an example of a mutation causing a very heterogeneous phenotype. It is the most frequent cause (80%) of the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), but it can also lead in addition or separately to type 2 diabetes, deafness, renal tubulopathy and/or cardiomyopathy. METHODS: To identify pathogenic processes induced by this mutation, we compared global gene expression levels of muscle biopsies from affected and unaffected mutation carriers with controls. RESULTS AND CONCLUSIONS: Gene expression changes were relatively subtle. In the asymptomatic group 200 transcripts were upregulated and 12 were downregulated, whereas in the symptomatic group 15 transcripts were upregulated and 52 were downregulated. In the asymptomatic group, oxidative phosphorylation (OXPHOS) complex I and IV genes were induced. Protein turnover and apoptosis were elevated, most likely due to the formation of dysfunctional and reactive oxygen species (ROS) damaged proteins. These processes returned to normal in symptomatic patients. Components of the complement system were upregulated in both groups, but the strongest in the symptomatic group, which might indicate muscle regeneration--most likely, protein damage and OXPHOS dysfunction stimulate repair (protein regeneration) and metabolic adaptation (OXPHOS). In asymptomatic individuals these processes suffice to prevent the occurrence of symptoms. However, in affected individuals the repair process terminates, presumably because of excessive damage, and switches to muscle regeneration, as indicated by a stronger complement activation. This switch leaves increasingly damaged tissue in place and muscle pathology becomes manifest. Therefore, the expression of complement components might be a marker for the severity and progression of MELAS clinical course.


Subject(s)
MELAS Syndrome/genetics , Point Mutation , RNA, Transfer, Leu/genetics , Adolescent , Adult , Aged , Apoptosis , Child , Child, Preschool , Complement Activation , Female , Gene Expression Profiling , Heterozygote , Humans , MELAS Syndrome/physiopathology , Male , Middle Aged , Muscle, Skeletal/physiopathology , Oxidative Phosphorylation , Proteins/metabolism , RNA, Transfer, Leu/metabolism
3.
Yeast ; 25(2): 129-40, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18081196

ABSTRACT

To extract functional information on genes and processes from large expression datasets, analysis methods are required that can computationally deal with these amounts of data, are tunable to specific research questions, and construct classifiers that are not overspecific to the dataset at hand. To satisfy these requirements, a stepwise procedure that combines elements from principal component analysis and discriminant analysis, was developed to specifically retrieve genes involved in processes of interest and classify samples based upon those genes. In a global expression dataset of 300 gene knock-outs in Saccharomyces cerevisiae, the procedure successfully classified samples with similar 'cellular component' Gene Ontology annotations of the knock-out gene by expression signatures of limited numbers of genes. The genes discriminating 'mitochondrion' from the other subgroups were evaluated in more detail. The thiamine pathway turned out to be one of the processes involved and was successfully evaluated in a logistic model to predict whether yeast knock-outs were mitochondrial or not. Further, this pathway is biologically related to the mitochondrial system. Hence, this strongly indicates that our approach is effective and efficient in extracting meaningful information from large microarray experiments and assigning functions to yet uncharacterized genes.


Subject(s)
Computational Biology/methods , Gene Expression Profiling , Genes, Mitochondrial/genetics , Genome, Fungal/genetics , Mutation/genetics , Saccharomyces cerevisiae/genetics , Thiamine/biosynthesis , Gene Expression Regulation, Fungal , Genes, Fungal/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
4.
Plant Mol Biol ; 29(3): 431-9, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8534843

ABSTRACT

Legume lectin stimulates infection of roots in the symbiosis between leguminous plants and bacteria of the genus Rhizobium. Introduction of the Pisum sativum lectin gene (psl) into white clover hairy roots enables heterologous infection and nodulation by the pea symbiont R. leguminosarum biovar viciae (R.l. viciae). Legume lectins contain a specific sugar-binding site. Here, we show that inoculation of white clover hairy roots co-transformed with a psl mutant encoding a non-sugar-binding lectin (PSL N125D) with R.l. viciae yielded only background pseudo-nodule formation, in contrast to the situation after transformation with wild type psl or with a psl mutant encoding sugar-binding PSL (PSL A126V). For every construct tested, nodulation by the homologous symbiont R.l. trifolii was normal. These results strongly suggest that (1) sugar-binding activity of PSL is necessary for infection of white clover hairy roots by R.l. viciae, and (2) the rhizobial ligand of host lectin is a sugar residue rather than a lipid.


Subject(s)
Lectins/physiology , Pisum sativum/genetics , Rhizobium leguminosarum/genetics , Base Sequence , Binding Sites , Cloning, Molecular , DNA Primers , Lectins/biosynthesis , Lectins/genetics , Molecular Sequence Data , Plant Lectins , Plant Roots , Plants , Plants, Genetically Modified , Plants, Toxic , Restriction Mapping , Nicotiana
5.
Glycoconj J ; 11(4): 375-80, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7873934

ABSTRACT

Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.


Subject(s)
Carbohydrate Metabolism , Lectins/metabolism , Mutagenesis, Site-Directed , Plant Lectins , Amino Acid Sequence , Binding Sites/genetics , Erythrocytes/drug effects , Hemagglutination Tests , Humans , Molecular Sequence Data , X-Ray Diffraction
6.
Plant Mol Biol ; 22(6): 1039-46, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8400124

ABSTRACT

Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability of Pisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val103) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (Tm) by some 10 degrees C in comparison with wild-type (wt) PSL, and the denaturation energy (delta H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe104) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugar-binding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced in Escherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 degrees C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are active in planta at ambient temperatures, but are inactive and possibly non-toxic at 37 degrees C in the intestines of mammals.


Subject(s)
Fabaceae/chemistry , Lectins/chemistry , Plants, Medicinal , Amino Acid Sequence , Amino Acids/chemistry , Calorimetry, Differential Scanning , Cloning, Molecular , Escherichia coli , Hemagglutination Tests , Immunoblotting , Lectins/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Plant Lectins , Protein Conformation , Sequence Homology, Amino Acid , Temperature
7.
Plant Mol Biol ; 20(6): 1049-58, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1463840

ABSTRACT

As part of a strategy to determine the precise role of pea (Pisum sativum) lectin, Psl, in nodulation of pea by Rhizobium leguminosarum, mutations were introduced into the genetic determinant for pea lectin by site-directed mutagenesis using PCR. Introduction of a specific mutation, N125D, into a central area of the sugar-binding site resulted in complete loss of binding of Psl to dextran as well as of mannose/glucose-sensitive haemagglutination activity. As a control, substitution of an adjacent residue, A126V, did not have any detectable influence on sugar-binding activity. Both mutants appeared to represent normal Psl dimers with a molecular mass of about 55 kDa, in which binding of Ca2+ and Mn2+ ions was not affected. These results demonstrate that the NHD2 group of Asn125 is essential in sugar binding by Psl. To our knowledge, Psl N125D is the first mutant legume lectin which is unable to bind sugar residues. This mutant could be useful in the identification of the potential role of the lectin in the recognition of homologous symbionts.


Subject(s)
Fabaceae/chemistry , Glucose/metabolism , Lectins/chemistry , Mannose/metabolism , Phytohemagglutinins/chemistry , Plants, Medicinal , Amino Acid Sequence , Asparagine/chemistry , Aspartic Acid/chemistry , Base Sequence , Binding Sites , Calcium/metabolism , DNA Mutational Analysis , Escherichia coli/genetics , Lectins/genetics , Manganese/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemistry , Phytohemagglutinins/genetics , Plant Lectins , Sequence Alignment , Structure-Activity Relationship
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