Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease.

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.

Chromatin structure analysis of the mouse Xist locus.

The Xist gene is expressed exclusively from the inactive X chromosome and plays a central role in regulating X chromosome inactivation. Here we describe experiments aimed at defining the extent of the active chromatin domain of the expressed Xist allele. By using an allele-specific general DNaseI sensitivity assay we show that there is preferential digestion of the expressed allele at sites within the transcribed locus but not in flanking sites located up to 70 kb 5'. A putative proximal boundary for the Xist domain is located within 10 kb upstream of promoter P1. Chromatin in the expressed domain was found to be acetylated at H4 in XX somatic cells but also in XY cells, where Xist is never expressed. A single clear exception to this was the Xist promoter, which is acetylated only in XX cells. These observations concur with the view that H4 acetylation may not be a general marker of active chromatin domains and further support data implicating local promoter acetylation as being of primary functional significance in vivo.

A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation.

We have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P1. This domain includes the promoter P0, which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation.

Physical mapping of 2000 kb of the mouse X chromosome in the vicinity of the Xist locus.

A physical map encompassing approximately 2.0 megabases (Mb) in the region of the mouse X-inactivation center has been constructed. The map extends from the Gjb-1 locus to the Xist locus and demonstrates the order of probes inseparable by genetic analysis. The deduced locus order is as follows: Gjb-1, Ccg-1, DXCrc171, Rps4, Phka, DXCrc177, DXCrc318, Xist. Detailed physical mapping in the region between the Phka and Xist loci indicates the position of CpG-rich islands associated with the 5' end of genes. The DXCrc177 and DXCrc318 loci, both defined by probes derived from linking clones, are associated with CpG-rich islands. The map provides a framework for the isolation of underlying sequences in the mouse X-inactivation center region.

The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus.

The Xist gene maps to the X inactivation center region in both mouse and human, and previous analysis of the 3' end of the gene has demonstrated inactive X-specific expression, suggesting a possible role in X inactivation. We have now analyzed the entire mouse Xist gene. The mature inactive X-specific transcript is 15 kb in length and contains no conserved ORF. The Xist sequence contains a number of regions comprised of tandem repeats. Comparison with the human XIST gene demonstrates significant conservation of sequence and gene structure. Xist RNA is not associated with the translational machinery of the cell and is located almost exclusively in the nucleus. Together with conservation of inactive X-specific expression, these findings support a role for Xist in X inactivation, possibly as a functional RNA or as a chromatin organizer region.

Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome.

X-chromosome inactivation in mammals is a regulatory phenomenon whereby one of the two X chromosomes in female cells is genetically inactivated, resulting in dosage compensation for X-linked genes between males and females. In both man and mouse, X-chromosome inactivation is thought to proceed from a single cis-acting switch region or inactivation centre (XIC/Xic). In the human, XIC has been mapped to band Xq13 (ref. 6) and in the mouse to band XD (ref. 7), and comparative mapping has shown that the XIC regions in the two species are syntenic. The recently described human XIST gene maps to the XIC region and seems to be expressed only from the inactive X chromosome. We report here that the mouse Xist gene maps to the Xic region of the mouse X chromosome and, using an interspecific Mus spretus/Mus musculus domesticus F1 hybrid mouse carrying the T(X;16)16H translocation, show that Xist is exclusively expressed from the inactive X chromosome. Conservation between man and mouse of chromosomal position and unique expression exclusively from the inactive X chromosome lends support to the hypothesis that XIST and its mouse homologue are involved in X-chromosome inactivation.

Abstract perceptual information for age level: a risk factor for maltreatment?

2 independent and geographically disparate samples show that physically abused 3-6-year-old children have cranial/facial proportions that are atypically older than their nonabused age-mates. 2 independent photographic samples corroborated this finding, 1 investigating 2-7-year-olds and 1 investigating 12-15-year olds. All 4 samples were blind rated by researchers who were unaware of the hypothesis. Comparisons are made with other physical variables associated with age level such as height. It is suggested that cranial/facial proportion is a particularly salient abstract specification for age level and may be used without awareness in caregiving decisions. This may lead to caregivers' unrealistic expectations of youngsters who appear atypically older than their chronological age. If these children do not meet these expectations, either as to how they should typically appear or typically behave, they may be more vulnerable to abuse. This possibility lends some support to Lorenz's idea that "infant-schema" properties such as physical proportions may be implicated in caregiving. However, it is asserted that cranial/facial information is simply a specification for age level, not a cause of abusive behavior.

The optical detection of platelets in plasma.

The attenuation of a light beam by a suspension of platelets in plasma was examined theoretically and experiments were performed to confirm this analysis. Interfering and modifying inputs were identified and their effects quantified. Recommendations for instrument design are made which minimise the effects of spurious inputs and enable platelet concentration in plasma to be estimated by simple optical methods. The most important recommendations are: (i) the use of light in the range 850--1000 nm so that water may be used instead of isologous platelet-free plasma as a blank and (ii) the emphasis on the need for the calibration of the system and the necessity for re-calibration if the optical system is disturbed or adjusted.

Preliminary observations on the technique of monitoring platelet aggregation in whole blood using a rotor system.

PLATELET aggregation is now recognised as an important early event in thrombosis. Problems associated with the measurement of the degree of platelet aggregation in plasma are outlined.An approach to the determination of the degree of platelet aggregation in whole blood using a new apparatus is described. When whole blood is placed in a circular transparent rotor chamber and subjected to rotational centrifugation at appropriate gravity forces the red cells are spun outwards leaving an inner zone of plasma containing single platelets. Since platelet aggregation does not occur in the absence of blood movement, the blood in the rotor is first agitated by discontinuous rotation using a suitable rotor drive system. Any platelet clumps which form are larger than single platelets and they may be later centrifuged out of the light path with the red cells. The clumps of platelets which have been removed from the plasma zone can be determined, by inference, by monitoring the plasma zone with a light beam. The precision in detecting platelet numbers may be improved by attention to factors such as the light path and wavelength.Because platelet concentrations, and perhaps plasma absorbance, vary in different individuals the light attenuation for blood plasma with and without platelets should be determined prior to estimating the degree of platelet aggregation under test conditions.

Details of a rotor system for investigating platelet aggregation in whole blood.

Working details of the prototype apparatus, for producing and monitoring platelet aggregation in whole blood, are described. The blood is subjected first to varying degrees of turbulence or agitation by the discontinuous rotation of a transparent cylindrical rotor chamber. During the period of turbulence platelets may aggregate under the influence of aggregating agents or of appropriate shear forces alone.The blood in the same chamber is subsequently subjected to continuous rotational centrifugation which leads to zonal separation of the plasma and platelets from the red cells by appropriate gravity forces. Any platelet clumps which have formed move outwards with the red cells leaving an inner zone of plasma through which a light beam is passed; optical measurements of the plasma zone indicate the number of unclumped platelets, since the formed aggregates have been centrifuged out of the light path. By comparison with the optical densities of platelet-free and platelet-rich plasma zones the degree of platelet aggregation which has taken place may be deduced.The technique of sampling different plasma zones for analysis by introducing a suitably shaped scoop into the zones of blood in the spinning chamber is described.The movement of blood and the rotor during discontinuous rotation has been analysed by high speed cinematography.