The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly(ADP-ribosyl)ation on core nucleosome structure

The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin was analyzed by gel electrophoresis, electron microscopy, and velocity sedimentation. In parallel, the interaction of automodified poly(ADP-ribose) polymerase with native and H1-depleted chromatin was analyzed. In H1-depleted chromatin histone H2B becomes the major poly(ADP-ribose) histone acceptor protein, whereas in native chromatin histone H1 was the major histone acceptor. Poly(ADP-ribosyl)ation of H1-depleted chromatin prevented the recondensation of polynucleosomes reconstituted with exogenous histone H1. This is probably due to the presence of modified poly(ADP-ribose) polymerase and hyper(ADP-ribosyl)ated histone H2B. Indeed, about 40% of the modified enzyme remained associated with H1-depleted chromatin, while less than 1% of the modified enzyme was bound to native chromatin. The influence of poly(ADP-ribosyl)ation on the chromatin conformation was also studied at the level of nucleosome in using monoclonal and polyclonal antibodies specific for individual histones and synthetic peptides of histones. In native chromatin incubated in the presence of Mg2+ there was a drop in the accessibility of histone epitopes to monoclonal and polyclonal antibodies whereas upon poly(ADP-ribosyl)ation their accessibility was found to remain even in the presence of Mg2+. In poly(ADP-ribosyl)ated H1-depleted chromatin an increased accessibility of some histone tails to antibodies was observed.     

Neonatal exposure to UVR alters skin immune system development, and suppresses immunity in adulthood

Neonates have a developing immune response, with a predisposition towards induction of tolerance. As the immune system develops, immunity rather than tolerance is induced, with this development of immunity occurring in response to external factors such as the environment. As ultraviolet radiation (UVR) suppresses immunity, it is likely that the effect of UVR on the neonatal immune system would be augmentation of the suppressive response. In support, childhood exposure to UVR has been linked with an increased incidence of melanoma; consistent with an increase in suppression. To address this, phenotypic and functional immune system studies were undertaken at 8 weeks after one single exposure of solar-simulated UVR to mice, when mice had reached adulthood. Subtle changes were observed in cell populations resident in the skin-draining lymph nodes (LNs) and there also appeared to be a subtle, but not statistically significant, increase in the production of interleukin-10 and interferon-γ. Importantly, these changes also corresponded with significant suppression of the contact hypersensitivity response in irradiated mice compared with their control counterparts. This suppression was apparent when antigen sensitisation occurred during the neonatal or adult period, and thus did not appear to be analogous to UVR-induced suppression in adults. Although the percentage of T regulatory cells was increased in the skin-draining LNs, they were induced in a different manner to those induced following adult UVR exposure, with no increase in function on a per-cell basis. It therefore appears that one single neonatal exposure to UVR alters development of the immune system, leading to long-term implications for induction of immunity.     

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.     

"Whey" proteins of milk of the red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroo

1. A comparison is made of gel electrophoretic patterns of the "whey" proteins of the milk of red (Macropus rufus) and eastern grey (Macropus giganteus) kangaroos at various stages of lactation. Qualitative and quantitative changes occur with time during the mature phase of lactation of both types. Their onset is related solely to the stage of lactation. "Whey" proteins are isolated and characterised and the nature of protein changes determined for the first time. 2. The anodic electrophoretic pattern is divided into 6 main zones (designated A F in order of decreasing mobility) and 2 cathodic zones (G and H) that are only detected in the milk of M. giganteus. 3. Zones A, B and C are milk specific. Zone B is present throughout lactation in both species and is an alpha-lactalbumin. Zones A and C are present only in late lactation, zone C, usually, but not always, appearing first. Zone A is an alpha-lactalbumin in M. giganteus, but is not an alpha-lactalbumin in M. rufus. Zone C appears to be the same protein in both species and is possibly a beta-lactoglobulin. 4. Zone D is kangaroo serum albumin and zone E is possibly a beta 2-microglobulin. Zone F contains three main iron (III) binding bands whose relative intensity varies with stage of lactation. Their intensity differs from the corresponding blood serum transferrin bands. 5. Zone H of Macropus giganteus is a lysozyme. 6. Lactose is present in the milk, but is not the principal sugar. 7. The significance of the results is discussed.     

Vasopressin and angiotensin induce inositol lipid breakdown in rat adenohypophysial cells in primary culture

Adenohypophysial cells from female Wistar rats were dispersed and maintained for 4 days in primary culture in the presence of [3H]myoinositol. The effects of several releasing hormones, corticotropin-releasing factor (CRF), arginine vasopressin (AVP), angiotensin II (A II), thyrotropin-releasing hormone (TRH), and luteinizing hormone-releasing hormone (LHRH) on the liberation of labelled inositol phosphate (InsP), inositol-bisphosphate (InsP2), and inositol-trisphosphate (InsP3) from prelabelled inositol lipids were tested alone and in combination. Of the corticotropin (ACTH) secretagogues tested, AVP and A II produced a dose-dependent increase in inositol phosphate accumulation. CRF was inactive. The ED50 values of about 1 nM for both AVP and A II were close to the corresponding dissociation constants for binding to pituitary membranes: and, in the case of A II, close to the ED50 for A II-induced inhibition of pituitary membrane adenylate cyclase. The responses to A II and AVP could be inhibited by [Sar1,Ile8]A II and the AVP antagonist d(Et2)-VAVP, respectively. The magnitude of the maximal effect of AVP on accumulation of inositol phosphates was small (25% increase over basal value) suggesting that this effect was restricted to a minor subpopulation of pituitary cells (probably corticotrophes). CRF did not potentiate AVP-induced inositol phosphates accumulation. Maximal A II-induced increase in inositol phosphates accumulation represented 150% of the basal value and was partially additive with that of TRH suggesting that lactotrophes represent the main A II-sensitive subpopulation.     

Accuracy of the Interpretation of Chest Radiographs for the Diagnosis of Paediatric Pneumonia

Introduction

World Health Organization (WHO) radiological classification remains an important entry criterion in epidemiological studies of pneumonia in children. We report inter-observer variability in the interpretation of 169 chest radiographs in children suspected of having pneumonia.

Methods

An 18-month prospective aetiological study of pneumonia was undertaken in Northern England. Chest radiographs were performed on eligible children aged ≤16 years with clinical features of pneumonia. The initial radiology report was compared with a subsequent assessment by a consultant cardiothoracic radiologist. Chest radiographic changes were categorised according to the WHO classification.

Results

There was significant disagreement (22%) between the first and second reports (kappa = 0.70, P<0.001), notably in those aged <5 years (26%, kappa = 0.66, P<0.001). The most frequent sources of disagreement were the reporting of patchy and perihilar changes.

Conclusion

This substantial inter-observer variability highlights the need for experts from different countries to create a consensus to review the radiological definition of pneumonia in children.     

Predominant Leptospiral Serogroups Circulating among Humans,Livestock and Wildlife in Katavi-Rukwa Ecosystem,Tanzania

BackgroundLeptospirosis is a worldwide zoonotic disease and a serious, under-reported public health problem, particularly in rural areas of Tanzania. In the Katavi-Rukwa ecosystem, humans, livestock and wildlife live in close proximity, which exposes them to the risk of a number of zoonotic infectious diseases, including leptospirosis.ConclusionsThe results of this study demonstrate that leptospiral antibodies are widely prevalent in humans, livestock and wildlife from the Katavi-Rukwa ecosystem. The disease poses a serious economic and public health threat in the study area. This epidemiological study provides information on circulating serogroups, which will be essential in designing intervention measures to reduce the risk of disease transmission.