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Biomedical subjects

D Segura

Publications and source records attributed to D Segura.

At least 19 recordsLinked to original sources
PubMed2005

Assessment of a platelet function analyser in horses: reference range and influence of a platelet aggregation inhibitor.

The objective of this study was to assess whether a new human platelet function analyser (the PFA-100) could be used to evaluate platelet function in horses and detect acetylsalicylic acid (ASA)-induced platelet dysfunctions. Citrated blood samples from 40 healthy horses were processed to obtain reference values for closure time (CT) using cartridges with collagen-ADP (CT-ADP) and collagen-epinephrine (CT-EPI) as platelet agonists. In addition, CT-ADP and CT-EPI were also measured before and 24 h after oral ASA administration in another 12 healthy horses. The sensitivity and specificity of the test were also determined. In normal horses, means+/-SD value for CT-ADP was 85.1+/-13.1 s (median, 82 s), and CT-EPI ranged from 158 to >300 s (median 291 s). Calculated reference ranges were 60.5-115.9 s and 158.5->300 s for CT-ADP and CT-EPI, respectively. Administration of ASA significantly (P<0.001) prolonged CT-ADP values from 91.0+/-13 to 113.5+/-14.4 s, and CT-EPI values were also significantly (P<0.008) prolonged after ASA administration. Sensitivity and specificity results for ADP cartridges showed that a prolonged CT value would be highly suggestive of a platelet aggregation inhibition. In conclusion, ADP cartridges can be used in horses to assess primary haemostasis and may be a valuable test for the detection of platelet aggregation inhibition.

Animals↗
PubMed2004

Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-beta-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium.

Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-beta-hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene ( pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly-beta-hydroxybutyrate.

Acetyl Coenzyme A↗
PubMed2004

Lower gastric ulcerogenic effect of suxibuzone compared to phenylbutazone when administered orally to horses.

The objective was to compare the gastrointestinal and general toxicity of suxibuzone (SBZ) to that of phenylbutazone (PBZ) when administered orally in horses. Fifteen healthy horses were allocated to three treatment groups. One group received a high dose of PBZ for two weeks; the second group was given an equimolecular dosage of SBZ; and a third group received placebo. Horses were daily monitored, and blood samples were collected before and during the study. On day 18, complete post-mortem examinations were performed. One horse treated with PBZ showed clinical signs of NSAID toxicosis. Small oral ulcers were also detected in other two horses from the PBZ group and in two horses from the SBZ group. There were no statistical differences in the blood parameters among groups. Ulcers in the stomach's glandular mucosa were observed in all horses of the PBZ group, while only two horses of the SBZ group showed ulcerations. PBZ horses had a significant higher ulcerated area, and gastric ulcers were significantly deeper than those in the SBZ and placebo groups. No other lesions were found in any other tissue. In conclusion, SBZ causes significantly lower gastric ulcerogenic effect than PBZ when administered orally at equimolecular doses in horses.

Animals↗
PubMed2003

Azotobacter vinelandii mutants that overproduce poly-beta-hydroxybutyrate or alginate.

Azotobacter vinelandii produces two polymers of industrial importance, i.e. alginate and poly-beta-hydroxybutyrate (PHB). Alginate synthesis constitutes a waste of substrate when seeking to optimize PHB production and, conversely, synthesis of PHB is undesirable when optimizing alginate production. In this study we evaluated the effect of a mutation in algA, the gene encoding the enzyme that catalyzes the first step of the alginate biosynthetic pathway in the production of PHB. We also evaluated production of alginate in strain AT6 carrying a phbB mutation that impairs PHB synthesis. The algA mutation prevented alginate production and increased PHB accumulation up to 5-fold, determined in milligrams per milligram of protein. Similarly, the phbB mutation increased alginate production up to 4-fold.

Acyltransferases↗
PubMed2002

Alginate production by an Azotobacter vinelandii mutant unable to produce alginate lyase.

Alginate is an industrially relevant linear copolymer composed of beta-1,4-linked D-mannuronic acid and its C-5 epimer L-guluronic acid. The rheological and gel-forming properties of alginates depend on the molecular weight and the relative content of the two monomers. Alginate produced by Azotobacter vinelandii was shown to be degraded towards the end of the culture, an undesirable situation in terms of potential alginate applications. A gene ( algL) encoding the alginate lyase activity AlgL is present within the alginate biosynthetic gene cluster of A. vinelandii. We constructed strain SML2, an A. vinelandii strain carrying a non-polar mutation within algL. No alginate lyase activity was detected in SML2. Under 3% dissolved oxygen tension, higher values of maximum mean molecular weight alginate were obtained (1240 kDa) with strain SML2, compared to those from the parental strain ATCC 9046 (680 kDa). These data indicate that AlgL activity causes the drop in the molecular weight of alginate produced by A. vinelandii.

Alginates↗
PubMed2002

Alginate production by Azotobacter vinelandii mutants altered in poly-beta-hydroxybutyrate and alginate biosynthesis.

Mutant AT268 of Azotobacter vinelandii - showing diminished production of poly-beta-hydroxybutyrate (PHB) due to a mutation in phbR (the gene coding for the transcriptional activator of the phbBAC biosynthetic operon); mutant CNT26, containing a mutation (muc26) that increases the transcription of gene algD (encoding GDP mannose dehydrogenase, the key enzyme in alginate biosynthesis); and mutant DM, carrying both phbR and muc26 mutations - were characterised in terms of alginate production, broth viscosity, and molecular weight of the alginate. All the mutants evaluated produced 25% less alginate with respect to that produced by the wild type. Unexpectedly and with no apparent relation to the phbR and muc26 mutations, mutant DM exhibited the highest molecular weight ever reported for a bacterial alginate (up to 4 x 10(6) Da), with a very low polydispersity index (1.3). Acetyl content in the alginate produced by this strain was low (1.4-2.6%). These characteristics make this mutant a very valuable source for producing alginates with improved properties.

Alginates↗
PubMed2001

Azotobacter vinelandii aldehyde dehydrogenase regulated by sigma(54): role in alcohol catabolism and encystment.

Encystment in Azotobacter vinelandii is induced by n-butanol or beta-hydroxybutyrate (BHB). We identified a gene, encoding an aldehyde dehydrogenase, that was named aldA. An aldA mutation impaired bacterial growth on n-butanol, ethanol, or hexanol as the sole carbon source. Expression of aldA increased in cells shifted from sucrose to n-butanol and was shown to be dependent on the alternative sigma(54) factor. A mutation in rpoN encoding the sigma(54) factor also impaired growth on alcohols. Encystment on n-butanol, but not on BHB, was impaired in aldA or rpoN mutants, indicating that n-butanol is not an inducer of encystment by itself but must be catabolized in order to induce encystment.

1-Butanol↗
PubMed2000

Beta-ketothiolase genes in Azotobacter vinelandii.

Azotobacter vinelandii is proposed to contain a single beta-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-beta-hydroxybutyrate (PHB) synthesis, and in beta-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953-963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial beta-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to beta-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to beta-hydroxyacyl-CoA and beta-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second beta-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on beta-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on beta-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced beta-ketothiolase activity and PHB accumulation, showing that this is the beta-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under beta-oxidation conditions and to possess beta-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for beta-ketothiolases in A. vinelandii.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

Molecular analysis of Spanish patients with AMP deaminase deficiency.

We found six patients with AMPD deficiency in muscle who were homozygous for the most common mutation, Q12X in the AMPD gene (AMPD1), associated with this disease. Three patients had AMPD deficiency alone, showing a mild clinical phenotype. Two patients showed a defect of PPL in muscle, and were homozygous for the most common mutation associated with McArdle's disease, R49X in the muscle PPL gene (PYGM). In one of these patients, the clinical phenotype was more severe than usually seen in patients with McArdle's disease. The remaining patient harbored the mtDNA A3243G mutation, showing one of the usual clinical patterns associated with this mutation. We conclude that the Q12X mutation in AMPD1 may result in a mild clinical effect; that it is frequent in the Spanish population, and therefore frequently associated with other metabolic diseases; and that the effect of the association of AMPD and PPL deficiencies seems to be neutral.

AMP Deaminase↗
PubMed1998

Myophosphorylase deficiency associated with a defect in complex I of the mitochondrial respiratory chain.

We studied a 21-year-old patient with clinical, biochemical and histochemical evidence of myophosphorylase deficiency and unusual repetitive episodes of pigmenturia. His muscle biopsy also revealed morphological signs of mitochondrial proliferation and a defect of complex I of the respiratory chain. His mother had exercise intolerance without myoglobinuria and no histochemical evidence of myophosphorylase deficiency. In muscle, the mother showed some ragged-red fibers, normal respiratory chain levels and a significant residual phosphorylase activity. Molecular genetic analysis revealed that the proband was homozygous for the mutation commonly found in McArdle's disease. The mother, father, and the five siblings were all heterozygous for the same mutation. Mitochondrial DNA analysis of the proband's muscle failed to demonstrate known mutations associated with his clinical pattern. Moreover, we sequenced his tRNA(Leu(UUR)) gene, a hot spot for mutations, showing no abnormality.

Adult↗
PubMed1998

Mutational inactivation of a gene homologous to Escherichia coli ptsP affects poly-beta-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii.

Strain DS988, an Azotobacter vinelandii mutant with a reduced capacity to accumulate poly-beta-hydroxybutyrate, was isolated after mini-Tn5 mutagenesis of the UW136 strain. Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to Escherichia coli ptsP which encodes enzyme INtr, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins. Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source. Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected. Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the ptsP mutation. The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen. Acetylene reduction activity but not transcription of the nitrogenase structural gene nifH was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose. The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration. These data suggest that a mutation in ptsP leads to a failure in poly-beta-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.

Amino Acid Sequence↗
PubMed1997

Association of genetically proven deficiencies of myophosphorylase and AMP deaminase: a second case of 'double trouble'.

We studied a 25-year-old man with paresis of the limbs and neck, scapular atrophy, facial weakness, exercise intolerance and frequent episodes of myoglobinuria. Muscle histochemistry and biochemistry revealed a combined defect of myophosphorylase and AMP deaminase. Molecular genetic analysis showed that the patient was homozygous for the two most common mutations associated with myophosphorylase and AMP deaminase deficiencies. This is the second documented case of genetic 'double trouble', which should be looked for in patients with unusual severe phenotypes.

AMP Deaminase↗
PubMed1996

Antiinflammatory activity of extracts from Aloe vera gel.

We studied the effects of aqueous, chloroform, and ethanol extracts of Aloe vera gel on carrageenan-induced edema in the rat paw, and neutrophil migration into the peritoneal cavity stimulated by carrageenan. We also studied the capacity of the aqueous extract to inhibit cyclooxygenase activity. The aqueous and chloroform extracts decreased the edema induced in the hind-paw and the number of neutrophils migrating into the peritoneal cavity, whereas the ethanol extract only decreased the number of neutrophils. The antiinflammatory agents indomethacin and dexamethasone also decreased carrageenan-induced edema and neutrophil migration. The aqueous extract inhibited prostaglandin E2 production from [14C]arachidonic acid. The chemical tests performed in the aqueous extract for anthraglycosides, reductor sugars and cardiotonic glycosides were positive. In the ethanol extract, the chemical tests performed for saponins, carbohydrates naftoquinones, sterols, triterpenoids and anthraquinones were also positive. In the chloroform extract, the chemical tests performed for sterols type delta 5, and anthraquinones were positive. These results demonstrated that the extracts of Aloe vera gel have antiinflammatory activity and suggested its inhibitory action on the arachidonic acid pathway via cyclooxygenase.

Aloe↗
PubMed1995

Clinical heterogeneity in two pedigrees with the 3243 bp tRNA(Leu(UUR)) mutation of mitochondrial DNA.

We studied two pedigrees with a mutation at the nucleotide 3243 of mitochondrial DNA (mtDNA). The proband from the first pedigree had clinically defined MELAS plus maternally transmitted insulin-dependent diabetes mellitus (IDDM). The propositus of the other pedigree had exercise intolerance, lactic acidosis and ragged-red fibers (RRF). In the first pedigree, both the mother and the sister's proband harbored the point mutation in their muscle. The mother had 40% of mutant mitochondrial genomes and the sister 70%. In the second pedigree, the mutation was present in both muscle and blood from the proband as well as in blood from all other members studied. Proportion of mutant mtDNA was 90% in muscle and ranged from 40% to 90% in blood.

Adult↗
PubMed1995

[Congenital myopathy with cores and nemaline rods in one family].

We present a mother and 2 children with congenital myopathy whose clinical signs were facial paresis in all three, and mild involvement of the lower extremities in the mother and one son. All three presented skeletal abnormalities, hypertelorism, arched palate, retraction of the Achilles tendon or short neck. Symptoms were not progressive and muscle biopsies showed central cores and nemaline rods in the mother and only nemaline rods in the 2 sons. The mother also suffered carpal tunnel syndrome, as had other members of the family as the result of autosomal dominant inheritance.

Adolescent↗
PubMed1995

[Adult onset mitochondrial myopathy without ophthalmoplegia. Four cases attributable to complex III and IV deficits in the respiratory chain].

Four adults with proximal myopathy of mitochondrial origin but no ocular involvement are presented. Biochemical analysis showed combined complex III and IV deficits in the respiratory chain in all cases, suggesting an apparent correlation between clinical phenotype and biochemical findings. Mitochondrial DNA analysis of muscle from 1 patient failed to detect either large-scale deletion or point mutations at position 3243 of tRNA(Leu(UUR)) or at 8344 of tRNA(Lys). The tissue specificity of the disease and the absence of family history suggest that a mutation in a nuclear DNA gene encoding a specific subunit of muscle could underlie this disease.

Adult↗