Histones and DNA methylation in mammalian chromatin. Differential inhibition by histone H1.

Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of double-stranded DNA (from Micrococcus luteus) by human placenta DNA methyltransferase. The absence of EDTA during the histone renaturation procedure abolished--at least in the 'physiological' range of the histones/DNA ratio--the inhibition. The H1 component was responsible for this inhibition, no effect being exerted by the other histones. H1 preparations were more effective if renatured in the presence of EDTA--90% inhibition being reached at a 0.3:1 (w/w) H1/DNA ratio. It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the histones, although this effect could also be shown with the 'inactive' H2a, H2b and H3 components, and was instead less evident with histone H1. The restriction to histone H1 of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin.

Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones.

After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid-soluble tightly-bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct H1-, H2A-, H2B-, H3- and H4-like components, the 'digested matrix' being however characterized by the absence of tightly-bound H1. These tightly-bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H1 components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H1 has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.

Inhibition of CpG methylation in linker DNA by H1 histone.

H1 exerts a specific in vitro inhibitory effect on enzymic DNA methylation. The experiments reported in this paper were undertaken in order to assess whether the lower methylation level found in internucleosomal DNA compared to core DNA is the in vivo consequence of the well-known localization of this histone in the linker region, as opposed to a possible deficiency of CpG dinucleotides in linker DNA. The methyl-accepting ability of H1-depleted oligonucleosomes from human placenta and of the corresponding core particles were assayed by addition of purified DNA methyltransferase, using S-adenosylmethionine as the methyl group donor. We have found that approx. 80% of newly-incorporated methyl groups are localized in linker DNA, which is indeed a good potential substrate for enzymic DNA methylation. Addition of quasi-physiological amounts of H1 to H1-depleted oligonucleosomes markedly reduced their methyl-accepting ability, while exerting a re-condensing effect on these particles, as revealed by the distortions of their circular dichroism spectra.

Autoimmunity to the GM2-1 islet ganglioside before and at the onset of type I diabetes.

Recently, the GM2-1 pancreatic islet ganglioside, proposed as a potential autoantigen in type I diabetes autoimmunity, has been biochemically characterized and found to be a novel ganglioside structure. In the present study, we aimed to determine whether an autoimmune response toward this novel islet molecule is 1) present in type I diabetes and is specifically directed against this molecule and not to gangliosides in general and 2) predictive of disease in high-risk subjects. To this end, the following patients have been studied: 1) 24 newly diagnosed type I diabetic subjects, 20 islet cell autoantibody (ICA)-negative first-degree relatives of type I diabetic subjects, and 25 age-matched normal control individuals; and 2) 31 prospectively evaluated ICA+ first-degree relatives of type I diabetic subjects who were followed for up to 10 years, during which 14 of them developed type I diabetes. A direct assay for autoantibodies to GM2-1 and to other pancreatic gangliosides (GM3, GD3, GD1a) was developed using an indirect immunoperoxidase technique performed directly on thin layer chromatography plates. Anti-GM2-1 autoantibodies (all belonging to the IgG class) were expressed in a high percentage of newly diagnosed type I diabetic subjects (71%), while no significant difference was found in the expression of antibodies directed against other pancreatic gangliosides (GM3, GD3, GD1a) among the different groups studied. Anti-GM2-1 autoantibodies were also present in ICA+ relatives (64%) (P < 0.001 vs. control subjects and ICA-relatives): in this group, life table analysis of progression to diabetes showed that anti-GM2-1 autoantibodies were significantly (P < 0.001) associated with disease, occurring in all relatives developing type I diabetes within 5 years and thus identifying a cohort of ICA+ subjects with markedly increased diabetes risk.

Does hypomethylation of linker DNA play a role in chromatin condensation.

The inhibitory effect that H1 histone exerts on the in vitro DNA methylation process, catalysed by mammalian DNA methyltransferase, together with the relative hypomethylation of linker DNA in eukaryotic cells chromatin, suggest that this hypomethylated state of linker DNA can be of importance in allowing or regulating H1-dependent chromatin condensation. In native oligonucleosomes (olnu), i.e., in chromatin fragments consisting of 5-20 nucleosomes each, there was a correlation between the effects of H1 on the DNA ellipticity at 280 nm and the in vitro assayed methyl-accepting ability. The same was true in H1-depleted or in H1-reconstituted preparations. Artificial methylation caused olnu DNA to lose its ability to allow cooperative H1-H1 interactions under ionic strength conditions similar to those known to affect the transition of the 10-nm filament to the 30-nm chromatin fiber. These results suggest that hypomethylation of linker DNA plays a role in the H1-H1 interactions that are needed for solenoid condensation.

Specific variants of H1 histone regulate CpG methylation in eukaryotic DNA.

Upon HPLC fractionation of human placenta or calf thymus H1 histone preparations, only some fractions enriched in the H1e-c variants were able to exert a severe inhibition on in vitro enzymatic DNA methylation. These fractions, though similar to the other variants in interacting with genomic DNA, were also the only ones which could bind CpG-rich ds-oligodeoxyribonucleotides (oligos). Both the 6-CpG ds-oligo and the DNA purified from chromatin fractions enriched in 'CpG islands' were good competitors for the binding of H1e-c to the 6meCpG ds-oligo. This ability to bind any DNA sequence and to suppress the enzymatic methylation in any sequence containing CpG dinucleotides suggests, for these particular H1 variants, a possible role in maintaining CpG island DNA and linker DNA at low methylation levels.

Poly(ADP-ribosyl)ation of proteins associated with nuclear matrix in rat testis.

We have previously demonstrated that a significant percentage of poly(ADPR) polymerase is present, as a tightly-bound form, at the third level of chromatin organisation defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [14C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix.

Inhibitors binding to L-aromatic amino acid decarboxylase.

The effect of a number of inhibitors of L-aromatic amino acid decarboxylase activity on the absorption spectrum of the enzyme-bound coenzyme has been studied. It has been observed that the compounds tested, even if devoid of the amino function and therefore unable to form the Schiff base with the coenzyme, modify significantly the enzyme spectrum, indicating their binding to the coenzyme active site. Spectral modifications suggest that at least two kinds of binding of inhibitors to L-aromatic amino acid decarboxylase may occur, depending on their structural features. Moreover, from the spectra obtained at different concentrations of the inhibitors their affinity constants have been determined: data indicate that the cathecol ring gives the largest contribution to the binding, while the presence of the carboxyl group, the aminic group and the aliphatic chain are responsible for a decrease in the binding, which could be relevant for the efficiency of the catalysis.

Persistence of azacytidine-induced SCEs and genomic methylation in CHO cells in vitro.

Many carcinogenic agents are able to affect the methylation level in mammalian cells cultivated in vitro. The capacity of azacytidine (AZA) to demethylate DNA can be used to examine the relationship between the genomic methylation level and cytogenetic end-points. Here we compared the sister-chromatid exchange (SCE) level with the genomic % methylcytosine in a Chinese hamster ovary cell line in vitro after giving a single 10-microM pulse of AZA. Both parameters were followed up to 16 cell cycles after the agent was removed. While the SCE level increased starting 2 cycles from the treatment and persisted for the entire 16 cycles, the methylcytosine level, after an initial 50% decrease, approached the control value, completely returning to it after 10 cell cycles. The possibility that the persistence in the SCE increase is an inherited phenomenon is discussed.

Glycoprotein distribution in non-histone chromatin proteins from pig liver.

Carbohydrate content of non-histone proteins from pig liver chromatin has been measured in different groups of chromatin fractions and does not seem to be related to the affinity of the proteins for DNA. Glycoproteins are preferentially located in the nuclease-sensitive fractions of chromatin. A 59,000 dalton glycoprotein has been identified as a characteristic components of a chromatin fraction solubilized by DNAase II.

64-slice MDCT arthrography in shoulder instability: our experience.

PURPOSE: This study was performed to assess the diagnostic accuracy of air-contrast 64-slice multidetector computed tomography (MDCT) arthrography in the evaluation of glenohumeral joint instability by comparison with conventional arthroscopy. MATERIALS AND METHODS: Fifty patients with a history of shoulder instability underwent MDCT arthrography with thin collimation scans. The raw data were transferred to a workstation and processed using multiplanar reformation (MPR) and volume rendering (VR) algorithms. All patients subsequently underwent conventional arthroscopy. The results of the two techniques were compared and their sensitivity and specificity calculated. RESULTS: We diagnosed eight anterosuperior labrum lesions (group 1), 32 anteroinferior labrum lesions (group 2) and two posterior labrum lesions (group 3). Overall sensitivity and specificity (groups 1, 2, 3) were 88% and 100%, respectively. In group 1, sensitivity was only 66% (four false negatives), whereas in groups 2 and 3, it was 94% (two false negatives) and 100%, respectively. The labrum lesions were also found to be associated, with 100% sensitivity and specificity, with 20 lax capsules, 17 Hill-Sachs lesions, five Bankart lesions, two Perthes lesions and three complete rotator-cuff tears. CONCLUSIONS: Air-contrast MDCT arthrography is fast, reproducible, well tolerated and very accurate in the evaluation of lesions causing shoulder instability.

[Lesions of the collateral ligaments of the ankle: diagnosis and follow-up with magnetic resonance and ultrasonography]. / Le lesioni dei legamenti collaterali del collo del piede: diagnosi e follow-up con risonanza magnetica ed ecografia.

Acute sprains are one of the most frequent ankle conditions; the lateral collateral ligaments are often involved. Currently, plain radiography and clinical examination are the methods of choice in the management of these injuries. This study was aimed at describing the MR findings of acute ankle sprains, to assess which ligaments are involved, to study the repair process during conservative treatment and, finally, to compare MR and US findings in acute and chronic injuries. We divided our study into a prospective and a retrospective parts. In the prospective study, MRI was performed in 20 consecutive patients with acute ankle sprain diagnosed at the emergency care unit of our institute and treated conservatively with braces. The patients with fractures were excluded. Follow-up was based on a series of MR exams performed every 30 days for 6 months. We diagnosed 18 injuries of the lateral collateral ligaments, 5 of the anterior talofibular ligament (ATFL) (2 partial and 3 complete tears) and 13 of both the ATFL and the calcaneofibular (CFL) (6 partial and 7 complete tears). The follow-up showed complete edema resorption at 30 days in 2 patients with no ligament injuries; the persistence of a medium-large amount of fluid in partial tears at 30 days and its complete resorption at 60 days; and, finally, the persistence of a medium-large amount of fluid at 180 days in 2 complete tears. No scar could be identified in all the patients with ligament injuries. The retrospective study was based on the comparison of MR (gold standard) and US findings in 78 patients with ankle sprain, 28 in the acute and 50 in the chronic phase. In the first group we found 9 ATFL injuries (6 partial and 3 complete tears), 5 ATFL and CFL injuries (3 partial and 2 complete tears), 2 complete tears of lateral collateral ligaments, 3 deltoid ligament (DL) injuries, 2 ATFL and DL injuries and 2 injuries of both lateral and medial collateral ligaments. US was in agreement with MRI in 85% of ATFL, 67% of CFL and 28% of DL injuries; US also yielded 2 false positives in PAA. In the second group of 50 patients, MRI showed 11 ATFL and 8 ATFL and CFL injuries, 3 injuries of lateral collateral ligaments, 6 DL and 8 ATFL, CFL and DL injuries and 5 complete tears of both internal and external collateral ligaments; 10 exams were negative. US had 58% agreement with MRI in ATFL, 46% in CFL and 21% in DL injuries. In this series, US yielded 3 false positives in PAA injuries.

Co-operative interactions of oligonucleosomal DNA with the H1e histone variant and its poly(ADP-ribosyl)ated isoform.

H1 histone somatic variants from L929 mouse fibroblasts were purified by reverse-phase HPLC. We analysed the ability of each H1 histone variant to allow the H1-H1 interactions that are essential for the formation of the higher levels of chromatin structure, and we investigated the role played by the poly(ADP-ribosyl)ation process. Cross-linking analysis showed that H1e is the only somatic variant which, when bound to DNA, is able to produce H1-H1 polymers; the size of polymers was decreased when H1e was enriched in its poly(ADP-ribosyl)ated isoform. Measurement of the methyl-accepting ability in native nuclei compared with nuclei in which poly(ADP-ribosyl)ation was induced showed that the poly(ADP-ribosyl)ated H1 histone had not been removed from linker regions, in spite of its different interaction with DNA.

Interactions of Epstein-Barr virus origins of replication with nuclear matrix in the latent and in the lytic phases of viral infection.

Eukaryotic DNA is organized into domains or loops generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions. The role of these regions in DNA replication is currently under investigation since they have been found in close association with origins of replication. Also, viral DNA sequences, containing the origins of replication, have been found attached to the nuclear matrix. To investigate the functional role of this binding we have studied, in Raji cells, the interaction between Epstein-Barr virus (EBV) origins of replication and the nuclear matrix in relation to the viral cycle of infection. We report here that both the latent (ori P) and the lytic (ori Lyt) EBV origins of replication are attached to the nuclear matrix, the first during the latent cycle of infection and the second after induction of the lytic cycle. These findings suggest that the binding of the origins of replication with the nuclear matrix modulates viral replication and expression in the two different phases of infection.

Does poly(ADP-ribosyl)ation regulate the DNA methylation pattern?

The existence of a possible correlation between poly(ADP-ribosyl)ation and DNA methylation processes was investigated. In vivo and in vitro experiments were carried out on L929 mouse fibroblasts preincubated for 24 h with or without 3-aminobenzamide, a well-known inhibitor of poly(ADP-ribose) polymerase. Both experimental approaches evidenced a close relationship between these two important nuclear enzymatic mechanisms, suggesting that the poly(ADP-ribosyl)ated isoform of H1 histone and/or long and branched protein-free ADP-ribose polymers could act as protecting agents against full methylation of the CpG dinucleotides in genomic DNA.

Some immunochemical properties of pig kidney DOPA decarboxylase.

Antibodies raised in rabbits against pig kidney DOPA decarboxylase show immunological cross-reactivity towards extracts from monkey, beef, rat and rabbit kidney. The influence of the immuno-reaction on the enzymatic activity has been investigated.

[CT-guided percutaneous treatment of osteoid osteoma]. / Trattamento percutaneo dell'osteoma osteoide sotto guida con Tomografia Computerizzata.

CT was used to localize and guide the percutaneous ablation of osteoid osteomas in 11 patients whose age ranged 5 to 63 years. The treatment was performed directly in the CT room ensuring maximum asepsis. General anesthesia was used in children and in vertebral and sacroiliac localizations, while the peripheral block was used in peripheral localizations. In the latter cases, an ischemic band was used to reduce bleeding. A dedicated drill resection system guided by a Kirschner guide wire was used for removal. The treatment was curative in the short period in all the patients, with complete symptom remission. Only one patient required retreatment after 6 months. In our series of patients no major complications, e.g., bleeding or infections, were observed. In 8 cases the resection yielded enough material for histology. To conclude, this technique can be considered a valuable alternative to surgery in the treatment of osteoid osteomas.