The Drosophila polycomb protein interacts with nucleosomal core particles In vitro via its repression domain.

The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with their cis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein-the Polycomb (PC) protein-with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

The glutamine-rich domain of the Drosophila GAGA factor is necessary for amyloid fibre formation in vitro, but not for chromatin remodelling.

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.

Cognitive impairment in a young marmoset reveals lateral ventriculomegaly and a mild hippocampal atrophy: a case report.

The number of studies that use the common marmoset (Callithrix jacchus) in various fields of neurosciences is increasing dramatically. In general, animals enter the study when their health status is considered satisfactory on the basis of classical clinical investigations. In behavioral studies, variations of score between individuals are frequently observed, some of them being considered as poor performers or outliers. Experimenters rarely consider the fact that it could be related to some brain anomaly. This raises the important issue of the reliability of such classical behavioral approaches without using complementary imaging, especially in animals lacking striking external clinical signs. Here we report the case of a young marmoset which presented a set of cognitive impairments in two different tasks compared to other age-matched animals. Brain imaging revealed a patent right lateral ventricular enlargement with a mild hippocampal atrophy. This abnormality could explain the cognitive impairments of this animal. Such a case points to the importance of complementing behavioral studies by imaging explorations to avoid experimental bias.

Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus.

In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.

Efficiency of Ber-EP4 antibody for isolating circulating epithelial tumor cells before RT-PCR detection.

We compared the efficacy of 4 methods for isolating circulating tumor cells: immunocapture with Ber-EP4-coated magnetic beads, density gradient separation, ammonium chloride, and distilled water-mediated erythrocyte lysis. Human blood from healthy volunteers was mixed with serial dilutions of prostate (LNCaP) and liver (HepG2) derived tumor cells. Isolation of circulating tumor cells was followed by reverse transcriptase-polymerase chain reaction with primers specific for prostate-specific antigen and alpha-fetoprotein. Ber-EP4 antigen expression was evaluated by immunohistochemistry in 27 hepatocellular carcinomas and 34 prostate adenocarcinomas. Peripheral blood samples from 12 patients with hepatocellular carcinoma and 10 with prostate adenocarcinoma also were tested. Density gradient separation and Ber-EP4 immunocapture were the most sensitive techniques for isolating circulating tumor cells in in vitro tests. Isolation by density gradient separation was significantly more sensitive than Ber-EP4 immunocapture when applied to peripheral blood samples of patients with cancer, a result consistent with the variable expression of Ber-EP4 antigen that we found by immunohistochemistry in prostate adenocarcinomas and hepatocellular carcinomas.

Quantification of free water in human dental enamel.

The amount of free water in 50 samples of air-dried enamel from permanent and deciduous teeth was measured by the Karl Fischer method. Samples included both contemporary and burial specimens. The mean values obtained showed that free water constituted about 1% of enamel mass. However, the range of individual values varied considerably, from 0.56 to 1.48%. The proportion of free water did not seem to depend on the patient's age, the type of tooth, or the relationship between the tooth and its oral environment. On the other hand, the deciduous enamel tested displayed a mean free water content that was three times the mean for the permanent teeth (3.01 vs. 1.00%) and the five burial teeth, a mean content of 1.68%.

A particularly aggressive combined glucagonoma and gastrinoma syndrome.

Duodeno-pancreatic biochemically polyfunctional endocrine tumour is a well known entity. Usually, only one hormone is responsible for the clinical features. We report a case of aggressive combined glucagonoma and gastrinoma tumour without metastases, causing respectively diabetic ketoacidosis and fulminant peptic ulcer, and death. Occasional patients can present with clinical features of both glucagonoma and gastrinoma. Diabetic patients exhibiting migratory skin lesions should be suspected of glucagonoma. In addition, a multidisciplinary approach to such patients including dermatologists, surgeons, radiologists and endoscopists is mandatory.

Minimal intervention dentistry: part 6. Caries inhibition by resin infiltration.

Resin infiltration has made possible an innovative way of treating initial carious lesions that fits perfectly with the concept of minimal intervention dentistry. Infiltration of carious lesions represents a new approach to the treatment of non-cavitated lesions of proximal and smooth surfaces of deciduous and permanent teeth. The major advantage of this method is that it is a non-invasive treatment, preserving tooth structure and that it can be achieved in a single visit. While this therapy can rightly be categorised as minimum intervention dentistry, clinical experience is limited and further controlled clinical trials are required to assess its long-term results. The inhibition of caries progression by resin infiltration should now be considered an alternative to invasive restorations, but involves early detection of lesions and does not allow for appropriate monitoring of the caries risk.

Minimal intervention dentistry: part 4. Detection and diagnosis of initial caries lesions.

The detection of carious lesions is focused on the identification of early mineral changes to allow the demineralisation process to be managed by non-invasive interventions. The methods recommended for clinical diagnosis of initial carious lesions are discussed and illustrated. These include the early detection of lesions, evaluation of the extent of the lesion and its state of activity and the establishment of appropriate monitoring. The place of modern tools, including those based on fluorescence, is discussed. These can help inform patients. They are also potentially useful in regular control visits to monitor the progression or regression of early lesions. A rigorous and systematic approach to caries diagnosis is essential to establish a care plan for the disease and to identify preventive measures based on more precise diagnosis and to reduce reliance on restorative measures.

In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication.

DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro.

[Characterization of liver regeneration in the albumin-urokinase transgenic mouse]. / Caractérisation de la régénération hépatique chez la souris transgénique albumine-urokinase.

UNLABELLED: Models of liver regeneration are essential to understand mechanisms of hepatic carcinogenesis, correct genetic diseases by gene transfer or hepatocyte transplantation. The expression in the liver of transgenic mice of a gene coding for a urokinase-type plasminogen activator (uPA mouse) induces hepatotoxicity and prolonged post-native liver regeneration from cellular clones which have inactivated the transgene. This model may have major applications but it remains necessary to characterize the liver regeneration pattern. METHODS: Histological and immunohistochemical studies of the liver of uPA and non-transgenic mice, 3, 7, 14, 21, 28, 42 and 56 days-old. Markers of cellular proliferation: 5-bromo-2'deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). RESULTS: Regenerative nodules were seen from day 14. These nodules then grew, became confluent and by 8 weeks constituted the entire liver mass. A semi-quantitative study of BrdU and PCNA showed a maximal labeling at day 7 (300 to 350 labeled cells/10 microscopic fields, mag 400). When the nodules appeared, 60 to 80% of the cells were labeled. The proportion of labeled cells decreased but was still greater than that observed in non transgenic mice up to day 56 (92 to 106 labeled cells vs 10 to 28, on day 28). CONCLUSIONS: In uPA mouse liver regeneration is significantly expanded, as compared to the regeneration following partial hepatectom. This study therefore has allowed to determine the best conditions for using this model.

Nucleosome movement by CHRAC and ISWI without disruption or trans-displacement of the histone octamer.

The chromatin accessibility complex (CHRAC) belongs to the class of nucleosome remodeling factors that increase the accessibility of nucleosomal DNA in an ATP-dependent manner. We found that CHRAC induces movements of intact histone octamers to neighboring DNA segments without facilitating their displacement to competing DNA or histone chaperones in trans. CHRAC-induced energy-dependent nucleosome sliding may, in principle, explain nucleosome remodeling, nucleosome positioning, and nucleosome spacing reactions known to be catalyzed by CHRAC. The catalytic core of CHRAC, the ATPase ISWI, also mobilized nucleosomes at the expense of energy. However, the directionality of the CHRAC- and ISWI-induced nucleosome movements differed drastically, indicating that the geometry of the native complex modulates the activity of its catalytic core.

Dual regulation of the Drosophila hsp26 promoter in vitro.

Efficient heat shock induction of Drosophila hsp26 gene transcription in vivo requires binding sites for heat shock factor (HSF) and GAGA factor (GAF) close to the TATA box (proximal elements) as well as 350 bp upstream of the start site of transcription (distal elements). We have evaluated the contribution of hsp26 promoter sequences to transcriptional activity in extracts from either heat shocked or unstressed fly embryos. Efficient transcription in either extract was governed by distinct regulatory principles. Transcription in extracts from unstressed embryos relied solely on GAGA elements which efficiently counteracted repression by abundant non-specific DNA-binding proteins. Transcription in extracts from heat shocked embryos depended only a little on GAGA elements, relying mainly on functional HSEs. Constitutively active recombinant HSF or native factor in an extract from heat shocked embryos was able to truly activate transcription essentially via proximal HSEs, but not when bound to distal sites. These two modes of regulation in vitro may correspond to the two functional states of the promoter before and after heat shock in vivo.