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1.
Redox Biol ; 20: 533-543, 2019 01.
Article in English | MEDLINE | ID: mdl-30508698

ABSTRACT

Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis.


Subject(s)
Cullin Proteins/metabolism , Mitochondria/metabolism , Proteasome Endopeptidase Complex/metabolism , Stress, Physiological , Cell Respiration , Mitochondria/genetics , Models, Biological , Reactive Oxygen Species/metabolism , Ubiquitin-Activating Enzymes/metabolism , Ubiquitination
2.
Bioresour Technol ; 218: 480-90, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27394994

ABSTRACT

Strain selection is one of the primary hurdles facing cost-effective microalgal biodiesel production. Indeed, the strain used affects both upstream and downstream biodiesel production processes. This study presents a screening procedure that considers the most significant criteria in microalgal biodiesel production including TAG production and wet extraction and recovery of TAGs. Fourteen freshwater and seawater strains were investigated. Large variation was observed between the strains in all the screening criteria. The overall screening procedure ultimately led to the identification of Parachlorella kessleri UTEX2229 and Nannochloropsis gaditana CCMP527 as the best freshwater and seawater strains, respectively. They featured the largest areal TAG productivity equal to 2.7×10(-3) and 2.3×10(-3)kgm(-2)d(-1), respectively. These two strains also displayed encouraging cell fragility in a high pressure bead milling process with 69% and 98% cell disruption at 1750bar making them remarkable strains for TAG extraction in wet environment.


Subject(s)
Biofuels , Chlorophyta/metabolism , Microalgae/metabolism , Photobioreactors , Stramenopiles/metabolism , Biomass , Fatty Acids/chemistry , Fresh Water , Lipids/chemistry , Nitrogen/chemistry , Pressure , Seawater
3.
Bioresour Technol ; 196: 339-46, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26253918

ABSTRACT

A study of cell disruption by bead milling for two microalgae, Nannochloropsis oculata and Porphyridium cruentum, was performed. Strains robustness was quantified by high-pressure disruption assays. The hydrodynamics in the bead mill grinding chamber was studied by Residence Time Distribution modeling. Operating parameters effects were analyzed and modeled in terms of stress intensities and stress number. RTD corresponded to a 2 CSTR in series model. First order kinetics cell disruption was modeled in consequence. Continuous bead milling was efficient for both strains disruption. SI-SN modeling was successfully adapted to microalgae. As predicted by high pressure assays, N. oculata was more resistant than P. cruentum. The critical stress intensity was twice more important for N. oculata than for P. cruentum. SI-SN modeling allows the determination of operating parameters minimizing energy consumption and gives a scalable approach to develop and optimize microalgal disruption by bead milling.


Subject(s)
Biotechnology/methods , Microalgae/cytology , Porphyridium/cytology , Stramenopiles/cytology , Biomass , Hydrodynamics , Microalgae/chemistry , Models, Theoretical , Porphyridium/chemistry , Pressure , Stramenopiles/chemistry
4.
Pharmacogenomics J ; 13(2): 189-96, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22064666

ABSTRACT

Erectile dysfunction (ED) is usually treated with sildenafil. Although genetic polymorphisms in the endothelial nitric oxide synthase (eNOS) gene may impair endogenous NO formation, there is little information about how eNOS polymorphisms and haplotypes affect the responses to sildenafil. We studied 118 patients; 63 patients had ED secondary to radical prostatectomy (PED) and 55 had organic, clinical ED. eNOS genotypes for three eNOS polymorphisms (T(-786)C, rs2070744; a variable number of tandem repeats (VNTR) in intron 4; and Glu298Asp, rs1799983) were determined, and eNOS haplotypes were estimated using PHASE 2.1. The clinical responses to sildenafil were evaluated and the patients were classified as good responders (GR) or poor responders (PR) when their changes in five-item version of International Index for Erectile Function questionnaire were above or below the median value. The TC/CC genotypes and the C allele for the T(-786)C polymorphism were more common in GR, compared with PR patients with PED. However, the 4b4a/4a4a genotypes and the 4a allele for the VNTR polymorphism in intron 4 were more common in GR, compared with PR patients with clinical ED. The C-4a-Glu haplotype was more common in GR than in PR patients with PED. Conversely, the T-4b-Asp haplotype was less common in GR than in PR patients with PED. No other significant differences were found. Our findings show evidence that eNOS polymorphisms affect the responses of PED and clinical ED patients to sildenafil.


Subject(s)
Erectile Dysfunction/drug therapy , Erectile Dysfunction/genetics , Nitric Oxide Synthase Type III/genetics , Piperazines/administration & dosage , Sulfones/administration & dosage , Adult , Aged , Aged, 80 and over , Biomarkers, Pharmacological , Erectile Dysfunction/pathology , Genotype , Haplotypes , Humans , Male , Middle Aged , Minisatellite Repeats , Piperazines/adverse effects , Polymorphism, Single Nucleotide , Prostatectomy , Purines/administration & dosage , Purines/adverse effects , Sildenafil Citrate , Sulfones/adverse effects , Surveys and Questionnaires
5.
Clin Rheumatol ; 22(2): 102-6, 2003 May.
Article in English | MEDLINE | ID: mdl-12740673

ABSTRACT

The aim of this study was to investigate whether interleukin-13 (IL-13) serum levels correlate to different nailfold capillaroscopy (NC) findings in patients with systemic sclerosis (SSc). IL-13 serum levels were measured using an ELISA method. The following NC abnormalities were considered: the presence of giant loops, haemorrhages, loss of capillaries, disorganisation of the vascular array, ramified/bushy capillaries and sludging of blood. A semiquantitative rating scale was adopted to score these changes, as well as a rating system for avascular areas and three morphological NC patterns ('early', 'active' and 'late'). Mean capillary density was determined by counting the total number of capillaries in a 1 mm length, and the arterial and venous diameters of the capillary as well as the total loop diameter were measured. In SSc patients IL-13 serum levels were significantly higher than in controls ( P < 00.1), whereas in patients with ( n=8) and without ( n=24) abnormal IL-13 serum levels (>17 pg/ml) the comparison of the NC features showed significantly relevant differences concerning a more frequent 'active' NC pattern ( P < 0.02), the presence of haemorrhages ( P < 0.0037) and sludging of blood ( P < 0.038), as well as larger total loop ( P < 0.036) and arterial ( P < 0.03) diameters, in those patients with elevated IL-13 serum levels. The study confirmed that IL-13 serum levels are higher in the sera of patients with SSc, and shows for the first time the significant correlations between this serological finding and some of the main relevant SSc capillaroscopic features, leading us to believe that this cytokine not only seems to sustain the immunological and fibrotic process of SSc, but might have a role in determining the more severe microvascular lesions in this disease.


Subject(s)
Interleukin-13/immunology , Microcirculation/physiopathology , Nails/blood supply , Scleroderma, Systemic/immunology , Vascular Diseases/immunology , Adult , Aged , Female , Humans , Interleukin-13/blood , Male , Microscopic Angioscopy , Middle Aged , Scleroderma, Systemic/blood , Scleroderma, Systemic/complications , Severity of Illness Index , Vascular Diseases/diagnosis , Vascular Diseases/etiology
6.
Clin Rheumatol ; 22(2): 107-11, 2003 May.
Article in English | MEDLINE | ID: mdl-12740674

ABSTRACT

The aim of the study was to evaluate whether the imbalance between IL-12 and IL-13 serum levels, reflecting Th1/Th2 activity, is related to class-specific circulating rheumatoid factors (RF) and anticardiolipin (aCL) antibodies in SLE. Using ELISA we measured serum IL-12, IL-13, RF and aCL antibodies in 73 SLE patients and 20 healthy controls. The determination of IL-12/IL-13 ratio showed that IL-12 levels were above (group A), equal to (group B) or below (group C) IL-13 levels in 71.2%, 15.1% and 13.7% of SLE patients, respectively. IgM-RF levels were significantly higher in group C than in groups A ( P < 0.002) and B ( P < 0.019). Group C had also higher IgM-aCL levels than group A ( P < 0.04). No relationship between IL-12/IL-13 ratio and clinical or other laboratory parameters was found. It was concluded that the increased levels of both IgM-RF and IgM-aCL in patients with prevalent Th2 activity suggest that the predominance of Th2 over Th1 could drive autoantibody production in SLE patients.


Subject(s)
Antibodies, Anticardiolipin/immunology , Interleukin-12/immunology , Interleukin-13/immunology , Lupus Erythematosus, Systemic/immunology , Rheumatoid Factor/immunology , Adolescent , Adult , Aged , Antibodies, Anticardiolipin/blood , Antibodies, Antiphospholipid/blood , Antibodies, Antiphospholipid/immunology , Female , Humans , Immunoglobulin M/blood , Immunoglobulin M/immunology , Interleukin-12/blood , Interleukin-13/blood , Lupus Erythematosus, Systemic/blood , Male , Middle Aged , Rheumatoid Factor/blood , Th1 Cells/immunology , Th2 Cells/immunology
7.
Gene ; 303: 63-8, 2003 Jan 16.
Article in English | MEDLINE | ID: mdl-12559567

ABSTRACT

We utilized the heat-sensitive mutant strain (Ts932), bearing a mutation at position 61 in the mitochondrial tRNA(Asp) gene, to identify nuclear genes involved in tRNA biogenesis; this mutant is defective in 3'-end processing and consequently in the production of mature mitochondrial tRNA(Asp). We transformed this strain with a yeast nuclear library and we isolated among other suppressors, an unknown, non-essential gene (called SMM1, corresponding to open reading frame YNR015w), which restored the growth on glycerol and a normal amount of processed tRNA(Asp) in the mutant. The gene contains a domain highly conserved in evolution from bacteria to human and its product has been recently shown to have dihydrouridine synthase activity.


Subject(s)
Cell Nucleus/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cell Nucleus/genetics , Conserved Sequence/genetics , DNA, Mitochondrial/genetics , Evolution, Molecular , Gene Expression Regulation, Fungal , Genetic Complementation Test , Mitochondria/genetics , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oxidoreductases , RNA, Transfer, Asp/genetics , RNA, Transfer, Asp/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Temperature
8.
EMBO Rep ; 4(1): 53-8, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12524521

ABSTRACT

We have taken advantage of the similarity between human and yeast (Saccharomyces cerevisiae) mitochondrial tRNA(Leu)(UUR), and of the possibility of transforming yeast mitochondria, to construct yeast mitochondrial mutations in the gene encoding tRNA(Leu)(UUR) equivalent to the human A3243G, C3256T and T3291C mutations that have been found in patients with the neurodegenerative disease MELAS (for mitochondrial 'myopathy, encephalopathy, lactic acidosis and stroke-like episodes'). The resulting yeast cells (bearing the equivalent mutations A14G, C26T and T69C) were defective for growth on respiratory substrates, exhibited an abnormal mitochondrial morphology, and accumulated mitochondrial DNA deletions at a very high rate, a trait characteristic of severe mitochondrial defects in protein synthesis. This effect was specific at least in the pathogenic mutation T69C, because when we introduced A or G instead of C, the respiratory defect was absent or very mild. All defective phenotypes returned to normal when the mutant cells were transformed by multicopy plasmids carrying the gene encoding the mitochondrial elongation factor EF-Tu. The ability to create and analyse such mutated strains and to select correcting genes should make yeast a good model for the study of tRNAs and their interacting partners and a practical tool for the study of pathological mutations and of tRNA sequence polymorphisms.


Subject(s)
Amino Acid Substitution , MELAS Syndrome/genetics , Mitochondria/physiology , Mutation, Missense , Peptide Elongation Factor Tu/physiology , Point Mutation , RNA, Fungal/genetics , RNA, Transfer, Leu/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , Biolistics , DNA, Mitochondrial/genetics , Gene Expression Regulation, Fungal , Genetic Vectors/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Elongation Factor Tu/genetics , Phenotype , Protein Biosynthesis , RNA, Fungal/chemistry , RNA, Transfer, Leu/chemistry , Recombinant Fusion Proteins/physiology , Saccharomyces cerevisiae/physiology , Sequence Alignment , Sequence Homology, Nucleic Acid
9.
Reumatismo ; 54(2): 113-21, 2002.
Article in Italian | MEDLINE | ID: mdl-12105679

ABSTRACT

OBJECTIVE: IL-12 is a proinflammatory cytokine produced by different antigen presenting cells. It has been shown to exert a critical role in inducing Th1 phenotype, thus initiating cell-mediated immune responses, but the significance of IL-12 in rheumatic diseases is not clear. Aim of the study was to determine IL-12 serum levels in immune rheumatic diseases and to analyse the relationship of this cytokine with main clinical and laboratory parameters. METHODS: We analysed, by ELISA, serum IL-12 levels in 114 patients with SLE, 47 with SS, 32 with SSc, 84 with RA, 138 with PA and in 17 healthy controls. We also examined main clinical and laboratory parameters, including autoantibody profile and clinical indices of disease activity. RESULTS: IL-12 serum levels were significantly higher in SLE and SS patients respect to controls. IL-12 serum levels were significantly higher in SLE patients compared to those affected by RA, PA and SSc. When we evaluated disease activity in SLE patients, we found significantly higher IL-12 serum levels in subjects with fever or in those without renal involvement, while no correlation was found in the other rheumatic immune diseases. CONCLUSIONS: These findings suggest that IL-12, modulating cell and humoral immune responses, is involved in the pathogenesis of immune rheumatic diseases, such as SLE and SS.


Subject(s)
Autoimmune Diseases/metabolism , Connective Tissue Diseases/metabolism , Interleukin-12/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Arthritis, Psoriatic/immunology , Arthritis, Psoriatic/metabolism , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Autoantibodies/blood , Autoimmune Diseases/immunology , Child , Connective Tissue Diseases/immunology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Interleukin-12/blood , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Male , Middle Aged , Organ Specificity , Scleroderma, Systemic/immunology , Scleroderma, Systemic/metabolism , Sjogren's Syndrome/immunology , Sjogren's Syndrome/metabolism , Th1 Cells/immunology
10.
Clin Exp Rheumatol ; 20(2): 213-6, 2002.
Article in English | MEDLINE | ID: mdl-12051401

ABSTRACT

OBJECTIVE: Several cytokines play a role in the production of autoantibodies such as RF and ANA by B-lymphocytes; the role of IL-13 in this process has not been previously studied. We investigated the relationship between the serum concentration of this cytokine and circulating autoantibodies. METHODS: IL-13 serum levels, as well as RF and ANA, were evaluated in 282 patients with autoimmune rheumatic diseases including RA (n=84), SLE (n= 114), SS (n=52) and Scl (n=32). RESULTS: Serum levels of IL-13 (pg/ml) were significantly higher in patients with RA (p < 0.00003), SLE (p < 0.03), SS (p < 0.0007), or Scl (p < 0.025) compared to controls. IL-13 serum levels correlated with those of RF in RA (p < 0.00001), SLE (p < 0.003) and Scl (p < 0.03). IL-13 levels were higher in RA (p<0.0003), SLE (p<0.005) and Scl (p<0.05) patients with RF than in patients without RF. SS patients with antiSSA/Ro antibodies had significantly higher IL-13 levels than SS patients without this autoantibody (p < 0.04). No statistically significant correlation was found between IL-13 levels and any other antinuclear autoantibody, total immunoglobulin levels or the main clinicalfeatures of each disease. CONCLUSION: The evidence of higher IL-13 levels in our RA, SLE, SS and Scl patients confirms that this cytokine is involved in the pathogenesis of autoimmune rheumatic diseases. The relationship of this cytokine with RF in RA, SLE and Scl, as well as with antiSSA/ Ro antibody in SS, strengthens the hypothesis that it plays a role in autoantibody production. However, the different autoantibody synthesis by B-cells recognises different pathways depending on the underlying autoimmune disease.


Subject(s)
Antibodies, Antinuclear/blood , Arthritis, Rheumatoid/immunology , Interleukin-13/blood , Lupus Erythematosus, Systemic/immunology , Scleroderma, Systemic/immunology , Sjogren's Syndrome/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Arthritis, Rheumatoid/blood , Female , Humans , Lupus Erythematosus, Systemic/blood , Male , Middle Aged , Scleroderma, Systemic/blood , Sjogren's Syndrome/blood
11.
Gene ; 286(1): 43-51, 2002 Mar 06.
Article in English | MEDLINE | ID: mdl-11943459

ABSTRACT

We have previously characterized a Saccharomyces cerevisiae mutant which contains a mutation in the essential rpn11/mpr1 gene coding for the proteasomal regulatory subunit Rpn11. The mpr1-1 mutation shows the phenotypic characteristics generally associated with proteasomal mutations, such as cell cycle defects and accumulation of polyubiquitinated proteins. However, for the first time, mitochondrial defects have also been found to be a consequence of a mutation in a proteasomal gene (Mol. Biol. Cell 9 (1998) 2917-2931). Since the mutant strain is thermosensitive both on glucose and on glycerol, we searched for revertants in order to shed light on the Rpn11/Mpr1 functions. Spontaneous revertants able to grow on glucose but not on glycerol at 36 degrees C were isolated, and, only from them, revertants able to grow at 36 degrees C on glycerol were selected. Revertants of the two classes were found to be extragenic. The detailed characterization of these extragenic suppressors demonstrates that the phenotypes related to cell cycle defects can be dissociated from those concerned with mitochondrial organization.


Subject(s)
Cell Cycle Proteins/genetics , Cysteine Endopeptidases/genetics , Endopeptidases , Mitochondria/genetics , Multienzyme Complexes/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cell Cycle/genetics , Microscopy, Confocal , Molecular Sequence Data , Mutation , Proteasome Endopeptidase Complex , Sequence Homology, Amino Acid , Suppression, Genetic/genetics
12.
Ann Rheum Dis ; 61(2): 174-6, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11796407

ABSTRACT

OBJECTIVES: To compare the pattern of interleukin (IL) 13 production in synovial fluid (SF) and serum of patients with psoriatic arthritis (PsA) with that in patients with rheumatoid arthritis (RA) and osteoarthritis (OA), investigating its relation to the proinflammatory cytokine IL12. METHODS: SF and serum IL13 levels were determined in 35 patients with PsA, 36 with RA, and 15 with OA. The main clinical and laboratory variables, including number of painful and/or swollen joints, Ritchie index, morning stiffness, erythrocyte sedimentation rate, level of C reactive protein, level of rheumatoid factor, and SF analysis, were also evaluated. RESULTS: SF IL13 levels were significantly higher in patients with PsA (p<0.02) or RA (p<0.012) than in patients with OA, with no significant difference between the former two. SF IL12 levels were significantly higher in patients with PsA (p<0.023) than in those with OA. Serum IL13 (p<0.0001) and IL12 (p<0.02) levels were lower in patients with PsA than in those affected by RA. Only patients with PsA had higher IL13 levels in SF than in serum (p<0.002). The IL13 SF/serum ratio was higher in the PsA group than in the group with RA (p<0.005) or OA (p<0.026). SF IL13 levels correlated with serum IL13 levels (p<0.0001) in RA and with SF IL12 levels (p<0.03) in PsA. CONCLUSIONS: In PsA, there appears to be localised production of IL13, in balance with IL12, in the inflamed joints. The distinct IL13 secretion profiles in PsA, RA, and OA may be related to the clinical pictures, reflecting the different pathogenic mechanisms involved in inflammatory and degenerative joint diseases.


Subject(s)
Arthritis, Psoriatic/immunology , Interleukin-13/analysis , Synovial Fluid/immunology , Arthritis, Psoriatic/blood , Arthritis, Psoriatic/pathology , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Blood Sedimentation , C-Reactive Protein/analysis , Chi-Square Distribution , Humans , Interleukin-12/analysis , Interleukin-12/blood , Interleukin-13/blood , Joints/pathology , Osteoarthritis/blood , Osteoarthritis/immunology , Osteoarthritis/pathology , Rheumatoid Factor/analysis
13.
Osteoporos Int ; 12(5): 412-6, 2001.
Article in English | MEDLINE | ID: mdl-11444091

ABSTRACT

Bone ultrasound parameters at the proximal phalanges of the hands were measured in 55 male patients with psoriatic arthritis (PA) (39 with peripheral radiologic involvement and 16 with axial involvement), comparing the findings with those in 16 rheumatoid arthritis (RA) patients, 20 ankylosing spondylitis (AS) patients and 55 age- and sex-matched normal controls. Mean values of amplitude-dependent speed of sound (Ad-SoS) and ultrasound bone profile score (UBPS) were significantly lower in RA (p < 0.001 and p < 1 x 10(-5)) and PA (p < 0.03 and p < 1 x 10(-6)) patients than in controls, while there was no statistically significant difference between AS patients and healthy subjects. Ultrasound parameters showed a significant negative correlation with age in all groups. In each patient group ultrasound values were unrelated either to disease duration or to inflammatory indices such as erythrocyte sedimentation rate and C-reactive protein. Moreover no significant differences were observed between ultrasound parameters of the dominant and the nondominant hand. PA patients with and without axial radiologic changes did not show any differences in ultrasound parameters. However, PA subjects with peripheral involvement only had significantly higher Ad-SoS (p < 0.04) and UBPS (p < 0.04) values than RA patients. PA patients with axial lesions had significantly lower (p < 0.04 and p < 0.01) ultrasound values than AS patients. These findings suggest that PA ultrasound techniques performed at the peripheral level are of value to speculate on bone involvement, although we think that ultrasound measurements cannot yet be recommended for monitoring bone involvement in these patients.


Subject(s)
Arthritis, Psoriatic/diagnostic imaging , Fingers/diagnostic imaging , Adult , Age Factors , Anthropometry , Arthritis, Psoriatic/physiopathology , Arthritis, Rheumatoid/diagnostic imaging , Arthritis, Rheumatoid/physiopathology , Bone Density , Humans , Male , Middle Aged , Spondylitis, Ankylosing/diagnostic imaging , Spondylitis, Ankylosing/physiopathology , Time Factors , Ultrasonography
14.
Yeast ; 18(3): 219-27, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11180455

ABSTRACT

We report the identification and characterization of a new mutation (ts9) in the Saccharomyces cerevisiae mitochondrial genome, which was first genetically mapped in the tRNAgly region and further identified by means of sequencing as consisting of a G to A transition at position 30 in the tRNA. The mutation causes an almost complete disappearance of mature tRNAgly, while a second mitochondrial mutation with a compensatory C to T change restores it in normal quantities; this points to the importance of the strong bond between bases 30 and 40 of the anticodon stem in the stabilization of the tRNA. In addition to resulting in a clear-cut heat-sensitive phenotype, the ts9 mutation creates a new EcoRV restriction site. Both properties were used as markers to monitor the successful (re) introduction of the mutated allele into a wild-type mitochondrial genome through biolistic transformation. The mutant frequency in the progeny as well as the correct integration of the mutated allele at its proper site demonstrate the feasibility of this method for creating and investigating specific mitochondrial tRNA mutations. The method will provide important applications for the use of yeast as a model system of human mitochondrial pathologies.


Subject(s)
Bacterial Proteins , RNA, Fungal/genetics , RNA, Transfer, Gly/genetics , RNA/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , Biolistics , Blotting, Northern , Blotting, Southern , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/genetics , DNA, Mitochondrial/physiology , Deoxyribonucleases, Type II Site-Specific/chemistry , Genome, Fungal , Hot Temperature , Humans , Mitochondria/genetics , Molecular Sequence Data , Neurodegenerative Diseases/genetics , Peptide Elongation Factor Tu , Point Mutation/physiology , RNA/chemistry , RNA, Mitochondrial , RNA, Transfer, Gly/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/growth & development , Sequence Analysis, DNA , Transformation, Genetic
15.
Lupus ; 9(1): 56-60, 2000.
Article in English | MEDLINE | ID: mdl-10713647

ABSTRACT

The relationship of rheumatoid factors (RF) with antiphospholipid syndrome (aPLS) and anticardiolipin antibodies (aCL) has rarely been investigated in systemic lupus erythematosus (SLE). We found IgM-RF, IgG-RF, IgA-RF, IgM-aCL, IgG-aCL, IgA-aCL, respectively, in 35.4%, 35.4%, 33.8%, 23.1%, 23.1%, 20.0% of 65 SLE patients. Class specific RFs were negatively associated (P<0.05) with IgG-aCL. The frequency of definite or probable aPLS according to Alarcon-Segovia classification criteria was significantly (P<0.05) different (8.7% vs 30.9%) in patients with or without IgG-RF. Among the other clinical features of SLE, we found that patients with IgG-RF, compared to patients lacking this autoantibody, showed a lower frequency (P<0.05) of serositis (21.7% vs 52.4%) and hematologic (52. 2% vs 80.9%) disorders. The levels of IgG-RF and IgM-RF negatively correlated with the number of ARA criteria (P<0.05) but not with the indices of diseases activity or damage. Our study shows that in SLE the presence of RFs are not markers of severity of the disease, but the negative association between IgG-RF and IgG-aCL suggests a distinct role of these autoantibodies in the pathology of SLE, whereas the presence of IgG isotype may identify a subset of SLE patients having a lower risk to develop some clinical manifestations such as aPLS.


Subject(s)
Antibodies, Anticardiolipin/analysis , Antiphospholipid Syndrome/immunology , Immunoglobulin Isotypes/analysis , Lupus Erythematosus, Systemic/immunology , Rheumatoid Factor/immunology , Adolescent , Adult , Aged , Antiphospholipid Syndrome/complications , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin Isotypes/classification , Lupus Erythematosus, Systemic/complications , Male , Middle Aged
16.
Mol Biol Cell ; 9(10): 2917-31, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9763452

ABSTRACT

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho degrees] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.


Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle/genetics , Cysteine Endopeptidases/genetics , DNA Replication , DNA, Fungal/biosynthesis , DNA, Mitochondrial/biosynthesis , Endopeptidases , Genes, Fungal , Mitochondria/genetics , Multienzyme Complexes/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Cycle/physiology , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , DNA, Fungal/genetics , DNA, Mitochondrial/genetics , Genes, Essential , Genetic Complementation Test , Genotype , Hot Temperature , Humans , Mice , Mitochondria/ultrastructure , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Mutagenesis, Insertional , Phenotype , Plants/genetics , Proteasome Endopeptidase Complex , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid
17.
Rev Rhum Engl Ed ; 65(12): 751-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9923043

ABSTRACT

OBJECTIVE: To determine which of two instruments, the Health Assessment Questionnaire (HAQ) and the Arthritis Impact Measurement Scales (AIMS), was more closely correlated with the main parameters reflecting activity and severity of psoriatic arthritis. METHODS: Both instruments were administered to 72 consecutive patients with psoriatic arthritis. RESULTS: Global HAQ and AIMS scores were closely correlated with each other (rs = 0.747; P < 0.00001). AIMS physical function scales--namely physical activity, dexterity, social activity and activities of daily living--were moderately or closely correlated with the main clinical disease activity parameters, most notably morning stiffness of axial joints (rs = 0.271-0.551). Scales measuring psychological status yielded weaker correlations with disease activity parameters (rs = 0.241-0.277) and were also correlated with the visual analog scale score for skin lesion severity. Morning stiffness of peripheral joints was correlated only with two AIMS scales, namely pain (rs = 0.532) and activities of daily living (rs = 0.303). Severity of radiological damage of peripheral and axial joints was most closely correlated with the scales of physical function, most notably physical activity. The global and scale HAQ scores showed moderate to close correlations with the main clinical disease activity parameters, most notably morning stiffness of axial joints. The global HAQ score was also correlated with radiological carpal involvement and with the radiological severity of peripheral joint involvement, whereas only the arising and hygiene scales were (moderately) correlated with the radiological severity of spinal involvement. CONCLUSION: Although both the HAQ and the AIMS were useful in assessing health status in psoriatic arthritis patients, only the AIMS captured some of the effects of the skin lesions. Our data also suggest that the AIMS may be more effective than the HAQ for evaluating the effect of radiological lesions produced by psoriatic arthritis.


Subject(s)
Arthritis, Psoriatic/physiopathology , Arthritis, Psoriatic/psychology , Health Status Indicators , Severity of Illness Index , Surveys and Questionnaires , Activities of Daily Living , Adult , Aged , Female , Humans , Male , Middle Aged , Pain , Range of Motion, Articular/physiology , Self-Assessment
18.
Curr Genet ; 31(6): 494-6, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9211792

ABSTRACT

In an attempt to identify new nuclear genes involved in the synthesis and processing of mitochondrial tRNAs, we utilized a multicopy nuclear library to suppress the heat-sensitive phenotype of a Saccharomyces cerevisiae mitochondrial mutant strain. This strain (Ts 932) is defective in the 3'-end processing of the mitochondrial tRNAAsp transcript. The nuclear genes coding for the mitochondrial elongation factor Tuf M and for the mitochondrial aspartyl-tRNA synthetase have been found to restore the temperature-resistant phenotype and to correct the RNA processing defect. Suppression was effective even when the genes were present on a centromeric plasmid.


Subject(s)
Aspartate-tRNA Ligase/genetics , DNA, Mitochondrial/genetics , Gene Dosage , Peptide Elongation Factor Tu/genetics , RNA, Transfer, Asp/genetics , Saccharomyces cerevisiae Proteins , Blotting, Northern , Cell Division/genetics , DNA Transposable Elements , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Mutagenesis , Mutation , Oxidoreductases , Phenotype , Plasmids/genetics , RNA Processing, Post-Transcriptional , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Temperature
19.
Yeast ; 13(4): 369-72, 1997 Mar 30.
Article in English | MEDLINE | ID: mdl-9133741

ABSTRACT

We report the sequence analysis of a 10,531 bp DNA of Saccharomyces cerevisiae chromosome VII. This sequence contains five complete open reading frames (ORFs) potentially encoding proteins longer than 100 amino acids and incomplete ORF encoding for the 3' part of the GCN5 gene (Georgakopoulos and Thireos, 1992). ORFs G9160 and G9155 correspond to the genes ENO1 (Holland et al. 1981) and PUP2 (Gergatsou et al., 1992) respectively. ORF G9165 codes for a protein which shares significant homology with known proteins present in databases (see below). The translated sequence of ORF G9170 shows 88% identity to the 6-phosphogluconate dehydrogenase encoded by the gene 6PGD from S. cerevisiae present in the SwissProt data library (P38720). This indicates that G9170 might code for a second 6-phosphogluconate dehydrogenase. ORF G9175 codes for a putative new member of the mitochondrial carrier family. A hypothetical tRNAThr (TGT) is also present in position 6842-6913.


Subject(s)
Chromosomes, Fungal/genetics , Genes, Fungal/genetics , Open Reading Frames/genetics , RNA, Transfer, Thr/genetics , Saccharomyces cerevisiae/genetics , Molecular Sequence Data , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Amino Acid
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