Roberto Strom

The Dynamics of Myogenin Site-specific Demethylation Is Strongly Correlated with Its Expression and with Muscle Differentiation

The molecular mechanisms underlying the activation of tissue-specific genes have not yet been fully clarified. We analyzed the methylation status of specific CCGG sites in the 5′-flanking region and exon 1 of myogenin gene, a very important myogenic differentiation factor. We demonstrated a loss of methylation, at the onset of C2C12 muscle cell line differentiation, limited to the CCGG site of myogenin 5′-flanking region, which was strongly correlated with the transcriptional activation of this gene and with myogenic differentiation. The same CCGG site was also found to be hypomethylated, in vivo, in embryonic mouse muscle (a myogenin-expressing tissue), as opposed to nonmuscle (nonexpressing) tissues that had a fully methylated site. In a C2C12-derived clone with enhanced myogenic ability, demethylation occurred within 2 h of induction of differentiation, suggesting the involvement of some active demethylation mechanism(s) that occur in the absence of DNA replication. Exposure to drugs that inhibit DNA methylation by acting on the S-adenosylmethionine metabolism produced a further reduction, to a few minutes, in the duration of the demethylation dynamics. These effects suggest that the final site-specific DNA methylation pattern of tissue-specific genes is defined through a continuous, relatively fast interplay between active DNA demethylation and re-methylation mechanisms.

Enzyme defense against reactive oxygen derivatives. II. Erythrocytes and tumor cells

SummaryThe enzymatic destruction of oxidizing products produced during metabolic reduction of oxygen in the cell (such as singlet oxygen, H2O2 and OH radical) involves the concerted action of superoxide dismutase-which removes O2- and yields H2O2-and H2O2 removing enzymes such as catalase and glutathione peroxidase. A difference in distribution or ratio of these enzymes in various tissues may result in a different reactivity of oxygen radicals.It was found that in red blood cells superoxide dismutase and catalase are extracted in the same fraction as hemoglobin, while glutathione peroxidase appears to be “loosely” bound to the cellular structure. This suggests that in red blood cells catalase acts in series with superoxide dismutase against bursts of oxygen radicals formed from oxyhemoglobin, while glutathione & peroxidase may protect the cell membrane against low concentrations of H2O2. On the other hand, catalase activity is absent in various types of ascites tumor cells, while glutathione peroxidase and superoxide dismutase are found in the cytoplasm. However, the peroxidase/dismutase ratio is lower than in liver cells, and this may provide an explanation for the higher susceptibility of tumor cells to treatments likely to involve oxygen radicals.

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

The biochemical mechanism of selective heat sensitivity of cancer cells: III. Studies on lysosomes

Abstract Lysosomes from Novikoff hepatoma cells show, if compared to those from normal or regenerating liver cells, a higher lability, as measured by the rate of release of acid hydrolases. Moreover, in lysosomes from neoplastic cells, the release of the various enzymes at 38 and 43 °C follows a non-homogeneous pattern. Neither intracellular lysosomal damage by photolysis, nor intralysosomal inhibition of acid hydrolases by trypan blue modify the onset of inhibition of respiration of tumor cells by elevated temperatures. It is concluded that the specific heat sensitivity of tumor cells is partially reflected at the level of their lysosomal membranes, but that is probably not the primary mechanism of the cellular damage caused by heat.

The biochemical mechanism of selective heat sensitivity of cancer cells—IV. Inhibition of RNA synthesis

Abstract The mechanism by which exposure of ascites tumour cells to supranormal temperatures causes an irreversible inhibition of uridine incorporation into RNA was investigated. Heat-treated cells were still able to incorporate labeled nucleotides, and even nucleosides, into RNA, if these precursors were added at sufficiently high concentrations. The passive permeability of the cell membrane increased exponentially with temperature, but this increase was fully reversible. At variance with the results obtained with agents which increase cell permeability, or which inhibit nucleoside permeation, heat treatment of Ehrlich ascites cells did not modify the ability of labeled uridine to be metabolized by these cells. The inhibition of uridine incorporation following heat treatment cannot therefore be attributed to a damage at the cell membrane level; the experimental data can be tentatively accounted for by the hypothesis of a block in the maturation of pre-rRNA into rRNA, with reutilization of the pre-rRNA degradation products.

Regulation by cannabinoid receptors of anandamide transport across the blood-brain barrier and through other endothelial cells

The endocannabinoid anandamide (AEA) has many neurovascular activities. However, it is not yet clear how AEA can be metabolized at the neurovascular interface, and how it can move through the vascular and the cerebral compartments. The results reported in this article show that isolated bovine brain microvessels, an ex vivo model of the blood-brain barrier, have detectable levels of endogenous AEA and possess the biochemical machinery to bind and metabolize it, i.e. type-1 and type-2 cannabinoid receptors (CB1R and CB2R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase, and the AEA-synthesizing enzymes N-acyltransferase and N-acyl-phosphatidylethanolamines-specific phospholipase D. We also show that activation of CB1R enhances AMT activity through increased nitric oxide synthase (NOS) activity and subsequent increase of NO production. AMT activity is instead reduced by activation of CB2R, which inhibits NOS and NO release. In addition, binding experiments and immunoelectronmicroscopy demonstrate that different endothelial cells vary in the expression of CB1R and CB2R on the luminal and/or abluminal sides. The different localization of CBRs can lead to a diverse effect on AMT activity on the luminal and abluminal membranes, suggesting that the distribution of these receptors may drive AEA directional transport through the blood-brain barrier and other endothelial cells.

Early demethylation of non-CpG, CpC-rich, elements in the myogenin 5′-flanking region: A priming effect on the spreading of active demethylation?

The dynamic changes and structural patterns of DNA methylation of genes without CpG islands are poorly characterized. The relevance of CpG to the non-CpG methylation equilibrium in transcriptional repression is unknown. In this work, we analyzed the DNA methylation pattern of the 5’-flanking of the myogenin gene, a positive regulator of muscle differentiation with no CpG island and low CpG density, in both C2C12 muscle satellite cells and embryonic muscle. Embryonic brain was studied as a non-expressing tissue. High levels of both CpG and non-CpG methylation were observed in non-expressing experimental conditions. Both CpG and non-CpG methylation rapidly dropped during muscle differentiation and myogenin transcriptional activation, with an active demethylation dynamics. Non-CpG demethylation occurred more rapidly than CpG demethylation. Demethylation spread from initially highly methylated short CpC-rich elements to a virtually unmethylated status. These short elements have a high CpC content and density, share some motifs and largely coincide with putative recognition sequences of some differentiation-related transcription factors. Our findings point to a dynamically controlled equilibrium between CpG and non-CpG active demethylation in the transcriptional control of tissue-specific genes. The short CpC-rich elements are new structural features of the methylation machinery, whose functions may include priming the complete demethylation of a transcriptionally crucial DNA region.

Isolated Brain Microvessels as In Vitro Equivalents of the Blood‐Brain Barrier: Selective Removal by Collagenase of the A‐System of Neutral Amino Acid Transport

On treatment with collagenase, brain microvessels, together with several protein components, lose some enzymatic activities such as alkaline phosphatase and γ‐glutamyltranspeptidase, whereas no change occurs in the activities of 5′‐nucleotidase and glutamine synthetase. The energy‐requiring „A‐system” of polar neutral amino acid transport is also severely inactivated, whereas the L‐system for the facilitated exchange of branched chain and aromatic amino acids is preserved. In the collagenase‐digested microvessels, this leads to loss of the transtimulation effect of glutamine on the transport of large neutral amino acids, because such transtimulation is due to a cooperation between the A‐ and L‐systems. By contrast, NH4+ maintains (and even enhances) its ability to stimulate the L‐system of amino acid transport, presumably through glutamine synthesis within the endothelial cells.

Expression of receptors for native and chemically modified low-density lipoproteins in brain microvessels

Despite the importance of cholesterol metabolism in the central nervous system, only relatively few studies have dealt with the cerebral uptake and transport of lipids into the brain compartment. These functions are mediated by the endothelium of brain microvessels, which forms the anatomical basis of the blood‐brain barrier. By a reverse transcriptase PCR study of messenger RNA expression we could show, in bovine brain microvessels, the presence of transcripts of native low‐density lipoprotein receptor and of both type I and II scavenger receptors. Brain microvessels therefore appear to play an active role in the uptake of native and modified low‐density lipoproteins.

Octopamine plasma levels and hepatic encephalopathy: a re-appraisal of the problem

An investigation on the blood levels of octopamine was carried out on 70 adult individuals. There was a statistically significant correlation between the levels of octopamine and hepatic encephalopathy. Normal subjects had values below 1 ng/ml, while patients with grade 3 or grade 4 encephalopathy constantly showed values above 3.2 ng/ml. In these two groups the distribution was fairly homogeneous. Through the differences between cirrhotics without neurologic involvement and those with grade 1 or 2 hepatic encephalopathy displayed statistical significance, distribution of values in these groups was rather non-homogeneous. Octopamine levels paralleled variations in mental state in 3 out 4 cases. No difference was found between venous and arterial values. The reaction of transmethylation used in the assay of octopamine was constantly found to be inhibited by the presence of plasma. This inhibition is probably due to the presence of one or more beta-hydroxyphenylethanolamines other than octopamine.