Paola Arslan

Apoptosis, DNA damage and ubiquitin expression in normal and mdx muscle fibers after exercise

The current view indicates that after eccentric exercise myofibers are mechanically damaged and therefore an inflammatory and necrotic process occurs. In the present paper we examine the possibility that apoptosis plays a role in normal and dystrophin‐deficient muscles after running. We analysed for apoptosis normal and dystrophin‐deficient mouse muscles after a night of spontaneous wheel‐running followed by two days of rest. Terminal deoxynucleotidyl transferase‐mediated endlabeling of DNA in nuclei in tissue sections and gel electrophoresis of extracted DNA showed the presence of fragmented DNA. Furthermore, ubiquitin, a protein whose appearance is related to apoptosis, increased in muscles of both dystrophic and normal runner mice. The present findings which confirm that DNA damage is absent in muscles of sedentary mice but present in muscles of runner mice offer a new hypothesis on early events of muscle damage.

Nuclear membrane-staining antinuclear antibody in patients with primary biliary cirrhosis.

An antinuclear antibody specific for nuclear membrane (ANMA) was observed by the immunofluorescence method in sera from patients with primary biliary cirrhosis (PBC). ANMA was present in 18 of 63 PBC sera (28.5) and in 1 of 431 control sera (0.2%). Its reaction appeared as a thin fluorescent ring confined to the nuclear envelope and was more evident when the sera were highly diluted and the fluorescence, due to frequently associated antimitochondrial antibody, faded. The ANMA fluorescent pattern was confirmed by indirect immunoperoxidase staining. ANMA was seen on both tissue cryostat sections and HEp-2 cells. It was a poorly or non-complement-fixing IgG, specific for an antigen resistant to DNase I, RNase, and trypsin. The significance of its presence in PBC in unknown at present. Identification of its antigen with one of the centromeric antigens is suggested.

Proton electrochemical gradient and phosphate potential in submitochondrial particles

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.

Pharmacokinetics and tumor concentration of intraarterial and intravenous cisplatin in patients with head and neck squamous cancer.

SummaryTumor-tissue platinum levels and major pharmacokinetic parameters were determined in 11 patients with head and neck squamous cancer (HNSC) who were given cisplatin (50 mg/m2 daily x 2 days) and 5-fluorouracil (5-FU; 1000 mg/m2, continuous infusion x 5 days) either i.a. or i.v. The plasma peak platinum concentrations (cmax) and the areas under the curve for total platinum concentration versus time (AUC) during i.a. infusions were lower than the i.v.cmax (mean, 1.92±0.28 and 4.08±2.80 mg/l, for i.a. and i.v. infusions, respectively) and AUC values (mean, 22.55±4.96 and 40.66±10.71 mg h−1 l−1 for i.a. and i.v. treatment, respectively), suggesting a first-passage extraction of the drug by the tumor mass during i.a. infusion. However, no statistically significant difference was found in platinum tumor concentrations after i.a. administration versus i.v. infusion. The lack of a difference in tumor platinum concentrations between the i.a. and the i.v. administration routes might be explained either by a relatively high blood supply to the tumor area, enabling efflux of the surplus free platinum from the tissue, or by the delay between drug infusion and biopsy. After three cycles of i.a. treatment good tumor remission was obtained with minimal local toxicity. Larger clinical studies testing the advantages of the i.a. administration route over i.v. infusion appear to be necessary.

Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage

Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury. In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h). Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation. On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology. Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD. A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001). Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained. Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.

Effects of chromium(VI) and chromium(III) on energy charge and oxygen consumption in rat thymocytes.

The cytotoxic effects of chromium compounds in two oxidation states have been studied in rat thymocytes. endogenous nucleotide levels and oxygen consumption were examined as relevant parameters of the physiological state of the cell. Incubation of rat thymocytes with Cr(VI) produced a marked unbalance of endogenous purine nucleotide pool and a parallel decrease in oxygen consumption. A close correlation between the reduction of oxygen consumption and ATP level in rat thymocytes treated with increasing concentrations of Cr(VI) has been found. In rat thymocytes permeabilized with digitonin and in isolated rat liver mitochondria both Cr(VI) and Cr(III) showed, at different range of concentrations, a marked inhibition of maximal oxygen consumption rate (uncoupled respiration). The effects observed were depending on chromium oxidation state and on different mitochondrial sites of substrate oxidation.

Oxidative metabolism of dopamine: A colour reaction from human midbrain analysed by mass spectrometry

In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.

Inhibition of FasL sustains phagocytic cells and delays myogenesis in regenerating muscle fibers

Macrophage‐muscle cell interactions are complex, and the majority is unknown. The persistence of inflammatory cells in skeletal muscle could be critical for myofiber viability. In the present paper, we show that FasL plays a role in the resolution of muscle inflammation. We analyzed inflamed muscles of normal mice treated from day 3 to day 8 with a FasL inhibitor (Fas‐Ig) or with control Ig. Treated muscles were collected at 3, 5, and 10 days. The treatment with recombinant Fas‐Ig protein induced a severe persistence of inflammatory cells at 5 days (115,000±27,838 vs. 41,661±6848, p<0.01) and 10 days from injury (145,500±40,850 vs. 5000±1000, p<0.001). Myofiber regeneration was highly impaired (37±14 vs. 252±28, p<0.01). Apoptosis of phagocytic cells was absent during Fas‐Ig treatment (0.9±0.6 vs. 1300±150,p<0.0001), but apoptotic, mononucleated cells appeared at day 10, 2 days after the suspension of Fas‐Ig administration. The time course of FasL expression during muscle inflammation, at mRNA and protein level, reveals a peak during myoblast proliferation. The peak of FasL expression coincides with the peak of apoptosis of phagocytic cells. In situ hybridization shows the co‐expression of FasL and MyoD mRNA in mononucleated cells, i.e., myoblasts. Experiments on the myoblast cell culture confirmed the expression of FasL in myoblasts. The findings shown here indicate one of the pathways to control myoblast‐macrophage interaction and might be relevant for the control of inflammatory cells in muscle tissue. Perhaps altering FasL expression with recombinant proteins could ameliorate inflammation in degenerative myopathies and up‐regulate muscle regeneration.

Dark and photohemolysis of erythrocytes by furocoumarins.

Abstract It has been shown that various furocoumarins are able to cause dark hemolysis in red blood cells (RBC). However, this effect is evident only at relatively high furocoumarin concentrations (4.6 x 10-4 M) - much higher than those used in photosensitization experiments or photochemotherapeutic treatments. Among the various furocoumarins examined in this study, only psoralen (Ps) and 3-carbethoxypsoralen (3-CPs) showed strong photohemolytic effects, while the other compounds revealed little or no activity. This fact indicates that Ps and 3-CPs are able to induce selective damage to the cell membrane of RBC. By pre-irradiating furocoumarin in ethanol or isotonic saline solutions and adding the irradiated solutions to a RBC suspension, hemolysis was observed in various compounds. The products of photolysis which form during pre-irradiation may be responsible, in terms of hemolysis, for toxic effects on RBC.

Effect of monovalent cation ionophores on lymphocyte cellular metabolism

The effect of valinomycin, nigericin and gramicidin on the cellular O2 consumption and on ATP content has been investigation. It has been found that while valinomycin and nigericin interfere with mitochondrial functions, gramicidin D does not show any appreciable effect. These results are explained in terms of the differing abilities of ionophores to redistribute among intracellular membranes.