Search PubMed⌕ Search

Biomedical subjects

M Contreras

Publications and source records attributed to M Contreras.

At least 181 records · Page 10Linked to original sources
PubMed1990

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.

Animals↗
PubMed1990

Screening plasma donors for high-titre antibody to cytomegalovirus using a latex agglutination test.

Cytomegalovirus (CMV) can cause severe morbidity in immunosuppressed patients. Regional transfusion centres in the UK are required to supply high-titre anti-CMV plasma to the Blood Products Laboratory (BPL), now called 'Bio-Products Laboratory', for the production of specific intravenous immunoglobulin at the Protein Fractionation Centre in Scotland. For this purpose, 703 plasmapheresis donors were screened by a modified latex agglutination test to assess their suitability as donors with high-titre anti-CMV. CMV antibodies were found in 48% of the donors. Seropositivity increased with age ranging from 33% in the 20- to 29-year age group to 57% in the 40-49 age group, although the 50-59 age group showed a slight decline to 53%. In all age ranges except the 40-49 group, seropositivity was highest in the female population. With the latex test, 14% of the total donors screened had a titre greater than or equal to 1 in 64, 7% greater than or equal to 1 in 128 and 4.2% greater than or equal to 1 in 256. Samples from 18 donors with titres greater than or equal to 1 in 128 were sent to BPL and all samples were found to have a sufficiently high titre for the production of CMV immune plasma. For BPL the 'cut-off' level for CMV immune plasma is a titre greater than or equal to 1 in 64 by complement fixation. Since the establishment of a panel of donors with high-titre anti-CMV plasma, more than 10 kg of plasma are dispatched monthly from our centre to BPL.

Adult↗
PubMed1990

Comparison of IgM and IgG anti-A and anti-B levels in Asian, Caucasian and Negro donors in the North West Thames Region.

This study was undertaken to test the widely held belief that higher levels of immune anti-A and anti-B are characteristic of Negro and Asian populations with a corresponding increased risk factor for AB0 haemolytic disease of the newborn. Overall, 300 serum samples from male and female Asian. Caucasian and Negro blood donors in the North West Thames Region of groups A, B and 0 were collected. The sera were titrated in microplates against pooled group A1 and pooled group B red blood cells. Although the results show that the highest levels for IgG anti-A and anti-B were found in group 0 female Negro donors, statistically these levels are not significantly higher than those of the other group 0 donors tested. We suggest that the potent anti-A and anti-B reported by others in Negro and Asian populations may arise from environmental rather than genetic factors.

ABO Blood-Group System↗
PubMed1990

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases.

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.

Adrenoleukodystrophy↗
PubMed1989

Adrenoleukodystrophy: impaired oxidation of fatty acids due to peroxisomal lignoceroyl-CoA ligase deficiency.

Very long chain fatty acids (lignoceric acid) are oxidized in peroxisomes and pathognomonic amounts of these fatty acids accumulate in X-adrenoleukodystrophy (X-ALD) due to a defect in their oxidation. However, in cellular homogenates from X-ALD cells, lignoceric acid is oxidized at a rate of 38% of control cells. Therefore, to identify the source of this residual activity we raised antibody to palmitoyl-CoA ligase and examined its effect on the activation and oxidation of palmitic and lignoceric acids in isolated peroxisomes from control and X-ALD fibroblasts. The normalization of peroxisomal lignoceric acid oxidation in the presence of exogenously added acyl-CoA ligases and along with the complete inhibition of activation and oxidation of palmitic and lignoceric acids in peroxisomes from X-ALD by antibody to palmitoyl-CoA ligase provides direct evidence that lignoceroyl-CoA ligase is deficient in X-ALD and demonstrates that the residual activity for the oxidation of lignoceric acid was derived from the activation of lignoceric acid by peroxisomal palmitoyl-CoA ligase. This antibody inhibited the activation and oxidation of palmitic acid but had little effect on these activities for lignoceric acid in peroxisomes from control cells. Furthermore, these data provide evidence that peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases are two different enzymes.

Adrenoleukodystrophy↗