A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Endemic goitre: a research protocol elaboration for eradication

The iodine deficiency (ID), which affects 1 person out of 6, is relatively neglected by the responsible of Public Health Service, particularly in developing countries. Consequences of ID are far from being negligible: mental retardation, hypofertility, hyperplasia, carcinoma, early ageing and, in very exposed areas, endemic cretinism. Nevertheless, eradication is easy and cheap but it requires rigorous protocols and control of results. The elaboration of these protocols is complex because it must be adapted to environment, population and financial possibilities of concerned countries. Based on our experience in this field, we propose a combined protocol, between the Public Health too liberal approach and that of too expensive research, which can be adapted to several situations.     

Immunological analysis of aconitase in pumpkin cotyledons: the absence of aconitase in glyoxysomes

Aconitase (EC 4.2.1.3) was purified by column chromatography and SDS-PAGE. Specific antibodies for aconitase were prepared after affinity purification of the antiserum with purified aconitase. The antibodies reacted with purified pumpkin aconitase, and with the 98 kDa protein band after electrophoretic fractionation of extracts of pumpkin cotyledons. Immunoblot analysis revealed a protein with similar molecular mass in extracts of several plants. The intensity of the 98 kDa band increased as pumpkin cotyledons developed in darkness, and decreased thereafter upon illumination. Aconitase activity showed a similar pattern. Anion exchange chromatography of a homogenate of pumpkin cotyledons, followed by western blotting, displayed the presence of immunoreactive protein bands only in fractions showing aconitase activity. The results indicate that the antibodies were specific for aconitase. When we investigated the presence of immunoreactive bands after sucrose gradient fractionation, aconitase was detected in the supernatant fractions and in mitochondria, while a very low amount was found in glyoxysomes. These data provide additional proof that aconitase is not localized in glyoxysomes.     

Discrimination between alpha-satellite DNA sequences from chromosomes 21 and 13 by using polymerase chain reaction.

alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.     

Purification and characterization of a novel pumpkin short-chain acyl-coenzyme A oxidase with structural similarity to acyl-coenzyme A dehydrogenases

A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs.     

Subclinical Myocardial Dysfunction and Cardiac Autonomic Dysregulation Are Closely Associated in Obese Children and Adolescents: The Potential Role of Insulin Resistance

Background

The prevalence of obesity is increasing among children/adolescents. Subtle cardiovascular abnormalities, responsible for a higher mortality later in life, have been reported in obese children/adolescents. The aims of the study were to evaluate cardiovascular autonomic regulation, by means of spectrum analysis of R-R interval variability, and myocardial function, by means of standard and tissue Doppler echocardiography, in a group of non-hypertensive asymptomatic obese children and adolescents; furthermore, the influence of insulin resistance was tested.

Subjects and Methods

R-R interval variability was analyzed during both the 70° head-up tilt and 24-hour electrocardiographic holter monitoring. Spectrum analysis of R-R interval variability provided the indices of sympathetic (low frequency [LF_RR]) and vagal (high frequency [HF_RR]) modulation of the sinoatrial node. Homeostasis model assessment of insulin resistance (HOMA-IR) was used to classify obese children/adolescents (n=72) as insulin resistant (n=37) and non-insulin resistant (n=35).

Results

In obese subjects: a) left ventricular mass was significantly (p<0.05) increased, whereas both the e/a ratio and the e''/a'' ratio were decreased; b) at rest, HF_RR was lower, and the LF_RR/HF_RR ratio was higher; c) during tilting, magnitude of tilt-induced inhibition of HF_RR was lower; d) during 24-hour electrocardiographic holter monitoring, LF_RR and the LF_RR/HF_RR ratio were higher, whereas HF_RR was lower; e) HOMA-IR inversely correlated with both the e''/a'' ratio (r=-0.655; p<0.001) and the tilt-induced LF_RR/HF_RR ratio (r=-0.933; p<0.001); and, f) the e''/a'' ratio correlated with the tilt-induced LF_RR/HF_RR ratio (r=0.501; p<0.001). Moreover, HF_RR at rest, magnitude of tilt-induced HF_RR reduction, and the e''/a'' ratio in insulin resistant obese children/adolescents were markedly lower when compared with the remaining subjects.

Conclusions

Subclinical abnormalities of myocardial function and of cardiac autonomic regulation were closely associated in obese children/adolescents and both correlated with the degree of insulin resistance.     

Effects of Nandrolone in the Counteraction of Skeletal Muscle Atrophy in a Mouse Model of Muscle Disuse: Molecular Biology and Functional Evaluation

Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.