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L Vergani

Publications and source records attributed to L Vergani.

At least 19 recordsLinked to original sources
PubMed2001

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI.

BACKGROUND: This study investigated the relationship between chromatin compactness, which is directly related to chromatin condensation, and DAPI uptake. Materials and Methods For the structural characterization of in situ chromatin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by permeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different time intervals after permeabilization. In order to compare nuclei of different sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chromatin condensation we introduced two new parameters, "average chromatin packing ratio" and "average dye spatial density." RESULTS: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin condensation induced by detergent were inversely related to modifications of nuclear volume. CONCLUSIONS: The combination of complementary information obtained by fluorescence microscopy, supported by a proper geometrical correction, and differential calorimetry allowed us to interpret the patterns of fluorescence intensities inside the nucleus in terms of chromatin structure.

Animals↗
PubMed2001

Changes of nuclear structure induced by increasing temperatures.

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.

Animals↗
PubMed2000

Rhabdomyosarcoma rho(0) cells: isolation and characterization of a mitochondrial DNA depleted cell line with 'muscle-like' properties.

Mutations of mitochondrial DNA are a significant cause of neuromuscular disease. Pathological mutant mitochondrial DNA has been studied in control nuclear backgrounds. These experiments entailed transfer of patient-derived mitochondria to rho(0) cells that lack mtDNA. A limitation of these studies has been the fact that the control nuclear backgrounds were unrelated to the affected tissues of patients. Therefore a rhabdomyosarcoma cell line that has 'muscle-like' properties was tested to determine whether it could be depleted of mtDNA. A human rhabdomyosarcoma cell line was treated with the DNA intercalating dye ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) for 45 days. The treatment induced complete and permanent loss of mitochondrial DNA (rho(0)) in the rhabdomyosarcoma cells, as mtDNA remained undetectable after 8 months of growth in medium without drug. Crucially, the rhabdomyosarcoma rho(0) cells retained the ability to differentiate into myotubes with expression of muscle specific isoenzymes. The rhabdomyosarcoma rho(0) cell line provides a model system for studying pathological mutant mtDNA in cells that more closely resemble human muscle than the hitherto available human rho(0) cell lines.

Antigens, Differentiation↗
PubMed1999

Changes of chromatin condensation in one patient with ataxia telangiectasia disorder: a structural study.

Differential scanning calorimetry and quantitative fluorescence microscopy have been employed to characterize the structure and organization of in situ chromatin in lymphoblastoid cells obtained from one ataxia telangiectasia (A-T) patient and one healthy family member. The proven capability of these biophysical techniques to measure changes of chromatin condensation directly inside the cells makes them very powerful in studying the eventual structural changes associated with the appearance of a pleiotropic genetic disorder such as ataxia telangiectasia. A-T syndrome is genetically heterogeneous and can be induced by different mutations of a single gene. The aim of this work is to determine whether the genetic mutation exhibited by the A-T patient of this study may be associated with modifications of chromatin structure and organization. Both the calorimetric and the fluorescence microscopy results acquired on cells from the A-T patient show that the structure and distribution of nuclear chromatin in situ change considerably with respect to the control. A significant decondensation of the nuclear chromatin is in fact associated with the appearance of the A-T disorder in the A-T patient under analysis, together with a rearrangement of the chromatin domains inside the nucleus.

Ataxia Telangiectasia↗
PubMed1999

Glycosaminoglycans treatment increases IGF-I muscle levels and counteracts motor neuron death: A novel nonanticoagulant action.

The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulin-like growth factor-I (IGF-I) content in the denervated muscles. EDL muscle denervation-induced atrophy in saline-treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 +/- 3 to 29 +/- 8. In GAGs-treated 42-day-old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 +/- 4. Treatment with GAGs selectively affects IGF-I content in denervated hindlimb muscles, which is augmented from 7.02 +/- 0.71 ng/mg tissue to 25.72 +/- 0.7 in the EDL and from 3.2 +/- 0.18 to a robust 211 +/- 9.6 in the soleus.

Animals↗
PubMed1999

Glycosaminoglycans boost insulin-like growth factor-I-promoted neuroprotection: blockade of motor neuron death in the wobbler mouse.

Wobbler mice display forelimb weakness, altered paw positioning, reduced running speed, muscle atrophy and motor neuron loss; co-treatment with glycosaminoglycans and insulin-like growth factor-I counteracts the progression of the disease. Reportedly, treatment with glycosaminoglycans or insulin-like growth factor-I slows the early stages of progressive forelimb dysfunction in wobbler mice. Our aim was to study whether the combination of these two drugs would result in greater neuroprotective effects. In a group of wobbler mice, combined treatment with daily s.c. administration of 20 microg/kg insulin-like growth factor-I and 1 mg/kg glycosaminoglycans was begun upon diagnosis at three weeks of age and continued for the next six weeks. This treatment halted motor neuron loss and markedly reduced the decay of forelimb muscle morphometry and function. Moreover, the mouse phenotype itself was strikingly improved. The effect of the combination treatment was significantly higher than that of the single drugs, even at a dosage as high as 1 mg/kg insulin-like growth factor-I. The ability of the insulin-like growth factor-I/glycosaminoglycans pharmacological cocktail to arrest the progression of motor neuron disease in wobbler mice and the safety of the low dose of insulin-like growth factor-I used hold promise that this combination might represent a novel approach for the treatment of motor neuron disease and peripheral neuropathies.

Aging↗
PubMed1999

Infantile lipid storage myopathy with nocturnal hypoventilation shows abnormal low-affinity muscle carnitine uptake in vitro.

An infant with respiratory insufficiency, cardiomyopathy, lipid storage myopathy and low muscle carnitine was diagnosed as having 'Ondine's curse' because of recurrent nocturnal hypoventilation. Carnitine uptake was studied in 20-day-old cultured muscle, where two distinct saturable transport components are recognized: the high- and low-affinity-uptake. Experimental evidence suggests that low-affinity-uptake is muscle-specific, operating at physiological carnitine concentration. In the patient's cultured myotubes, the low-affinity-uptake K(m) was 260% of controls (P < 0.01), whereas kinetic parameters of high-affinity uptake were normal. The high K(m) indicates an immature or altered carnitine muscle carrier, which may decrease the physiologic carnitine uptake.

Carnitine↗
PubMed1999

Riboflavin therapy. Biochemical heterogeneity in two adult lipid storage myopathies.

Two unrelated adult males, aged 36 (patient 1) and 25 (patient 2) years, presented with subacute carnitine-deficient lipid storage myopathy that was totally and partly responsive to riboflavin supplementation in the two patients, respectively. Plasma acyl-carnitine and urinary organic acid profiles indicated multiple acyl coenzyme A dehydrogenase deficiency, which was mild in patient 1 and severe in patient 2. The activities of short-chain and medium-chain acyl coenzyme A dehydrogenases in mitochondrial fractions were decreased, especially in patient 2. This was in agreement with Western blotting results. Flavin-dependent complexes I and II were studied by immunoblotting and densitometric quantification of two-dimensional electrophoresis with comparable results. Complex I was present in normal amounts in both patients, whereas complex II was decreased only in the pretherapy muscle of patient 2. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) concentrations in muscle and isolated mitochondria, and the activity of mitochondrial FAD pyrophosphatase, showed that patient 1 had low levels of FAD (46%) and FMN (49%) in mitochondria, with a significant increase (P < 0.01) in mitochondrial FAD pyrophosphatase (273%) compared with controls. Patient 2 had similar low levels of FAD and FMN in both total muscle (FAD and FMN 22% of controls) and mitochondria (FAD 26%; FMN 16%) and normal activity of mitochondrial FAD pyrophosphatase. All of these biochemical parameters were either totally or partly corrected after riboflavin therapy.

Adult↗
PubMed1999

Introduction of heteroplasmic mitochondrial DNA (mtDNA) from a patient with NARP into two human rho degrees cell lines is associated either with selection and maintenance of NARP mutant mtDNA or failure to maintain mtDNA.

Mitochondria from a patient heteroplasmic at nucleo-tide position 8993 of mitochondrial DNA (mtDNA) were introduced into two human tumour cell lines lacking mtDNA. The donor mitochondria contained between 85 and 95% 8993G:C mtDNA. All detectable mtDNA in the mitochondrially transformed cells contained the pathological 8993G:C mutation 3 months after transformation. These results suggest that 8993G:C mtDNA had a selective advantage over 8993T:A mtDNA in both lung carcinoma and osteo-sarcoma cell backgrounds. In contrast, two other presumed pathological mtDNA variants were lost in favour of 'wild-type' mtDNA molecules in the same lung carcinoma cell background. Taken together, these findings suggest that the transmission bias of mtDNA variants is dependent upon a combination of nuclear background and mtDNA genotype. A second phenomenon observed was a marked decrease in the growth rate of many putative transformed cell lines after 6 weeks of culturing in selective medium, and in these cell lines mtDNA was not readily detectable by Southern blotting. Restriction endonuclease analysis and sequencing of amplified mtDNA demonstrated that the slow growing cells contained little or no mtDNA. It is concluded that these cells represented transient mitochondrial transformants.

Ataxia↗
PubMed1998

Systemic administration of insulin-like growth factor decreases motor neuron cell death and promotes muscle reinnervation.

Neonatal sciatic nerve axotomy causes motoneuron death and muscle denervation atrophy. The aim of the present study was to determine whether insulin-like growth factor-I (IGF-I) administration promotes muscle reinnervation and counteracts motor neuron loss after such an injury. Six weeks after sciatic nerve axotomy performed in 2-day-old pups, the number of motor neurons, as assessed by retrograde transport of horseradish peroxidase injected into the extensor digitorum longus (EDL) muscle, was reduced from 52 +/- 3 to 26 +/- 3. Subsequent administration of IGF-I at the doses of 0.02 mg/kg or 1 mg/kg increased the number of motor neurons to 35 +/- 2 and 37 +/- 5, respectively. The effect on motoneuron survival was accompanied by improved muscle fibre morphometry and restoration of indirect EDL muscle isometric twitch tension, which was about 80 % of control values for both doses of IGF-I compared with 60% observed with saline treatment. Reinnervated EDL muscle from saline-treated rats cannot hold tetanic tension, which is, however, achieved after IGF-I treatment at either dose. Thus, both high and low doses of IGF-I counteracted motoneuron death and improved muscle reinnervation following neonatal sciatic nerve axotomy. IGF-I at 5 microg/kg failed to increase muscle reinnervation.

Animals↗
PubMed1998

Neuroprotection, neuroregeneration, and interaction with insulin-like growth factor-I: novel non-anticoagulant action of glycosaminoglycans.

We present recent developments in the area of glycosaminoglycans (GAGs) and their possible interaction with insulin-like growth factor-I (IGF-I). GAGs are constituents of proteoglycans, and the combination of a core protein and a specific GAG makes a unique proteoglycan with a precise developmental pattern and with the ability to bind growth factors. This process is apparently regulated by the moiety of the peripheral GAGs. The supplementation of GAGs promotes neuritogenesis in vitro and stimulates nerve regrowth and muscle reinnervation, an effect correlated with an increase in trophic factor mRNA expression. In the case of neonatal nerve lesion, there is in addition an enhanced motor neuron survival, accompanied by higher levels of IGF-I in plasma and denervated muscle. The neurotrophic and neuroregenerative effects of exogenous GAGs were also observed in motor neuron disease in the wobbler mouse.

Animals↗
PubMed1998

Muscle reinnervation following neonatal nerve crush. Interactive effects of glycosaminoglycans and insulin-like growth factor-I.

This study shows that glycosaminoglycans promote muscle reinnervation following neonatal sciatic nerve injury. Such an effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy. In addition glycosaminoglycan treatment of lesioned rats increased insulin-like growth factor-I messenger RNA and protein in the reinnervated muscle, and insulin-like growth factor-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of nerve lesioned rats with insulin-like growth factor-I promoted muscle reinnervation and prevention of muscle fibre atrophy, higher levels of insulin-like growth factor-I in the reinnervated muscle and of insulin-like growth factor-I and insulin-like growth factor binding proteins in plasma. These data suggest that glycosaminoglycans are potent stimulants of muscle reinnervation and that their effects may be mediated by increased levels of insulin-like growth factor-I.

Acetylcholinesterase↗
PubMed1998

X-ray small angle scattering study of chromatin as a function of fiber length.

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.

Animals↗
PubMed1998

Effects of polyamines on higher-order folding of in situ chromatin.

Modifications of the higher-order chromatin structure induced by polyamines have been quantitatively investigated in situ through a non-invasive biophysical approach using Differential Scanning Calorimetry and Quantitative Fluorescence Microscopy. Calorimetric and intensitometric profiles have been acquired for samples of native thymocytes, alternatively suspended in buffers, with or without natural polyamines (spermine and spermidine). The results here reported show that the structure and distribution of nuclear chromatin in situ considerably change upon the ionic composition of the environment. A quantitative analysis of this data and a comparison with previous results obtained from isolated chromatin fibers was carried out. Finally, an inverse relationship between chromatin condensation and nuclear volume was observed.

Animals↗
PubMed1997

Effects of low doses of glycosaminoglycans and insulin-like growth factor-I on motor neuron disease in wobbler mouse.

In this study we examined the effects of insulin-like growth factor-I (IGF-I) and of glycosaminoglycans (GAGs) on the progressive motor neuron disease in wobbler mice. After clinical diagnosis at age 3 weeks, mice received daily subcutaneous injections of IGF-I, or GAGs, or saline for 3 weeks. The histometric analysis revealed that biceps muscle fiber diameter was reduced in wobbler mice and that treatments with GAGs and IGF-I prevented such a drop. The number of atrophic small fibers was markedly reduced and that of the larger ones augmented. No effects on body growth and biceps muscle weight were observed. The combined AChE-silver staining revealed that both treatments promoted intramuscular axonal sprouting. The typical decline of grip strength in wobbler mice was also prevented. This study suggests that GAGs and IGF-I administrations can retard the onset of motor deficit, and reduce muscle atrophy in wobbler mice.

Acetylcholinesterase↗
PubMed1997

Effects of histone acetylation on chromatin structure.

The effect of histone acetylation was monitored on CHO chromatin structure, following the addition of 7 mM Na-butyrate to the cell culture medium. The properties of both control and hyperacetylated chromatins and nuclei were investigated by circular dichroism, ethidium bromide intercalation, differential scanning calorimetry, and affinity chromatography. Our results are compatible with modest but significant alterations in the various levels of chromatin organization, as a result of the charge neutralization of some lysine residues within the N-terminal region of the histonic octamer. Namely, large statistically significant differences do exist in the heat capacity thermograms of native nuclei, where unfolding into single nucleofilament of the highly packed native chromatin superfiber appears associated with acetylation; at the same time CD, EB, and affinity chromatography point to modest but consistent differences in the compactness of isolated nucleosomes and polynucleosomes.

Acetylation↗
PubMed1997

Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.

Blue native-polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi-subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin-responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.

Adenosine Triphosphatases↗