Paul T. Sharpe

Canine distemper virus localised in bone cells of patients with Paget's disease

The technique of in situ hybridisation was applied using radioactively labelled riboprobes to examine for the presence of canine distemper (CDV) and measles (MV) RNA in Pagets disease of bone. The results indicate that in 41% of Pagets patients, CDV RNA could be detected in osteoclasts, osteoblasts, and osteocytes, but not in controls. In contrast, RNA to the measles virus was not detected. We suggest that CDV may in some cases play a role in the aetiology of Pagets disease.

Canine distemper virus transcripts sequenced from pagetic bone

Pagets disease is a chronic disease of the skeleton which is believed to be caused by a persistent virus of the paramyxovirus family. There is still conflict as to the precise identity of the virus(es) involved in causing this disease. Our previous results using in situ hybridisation have implicated distemper as a possible cause of this disease. In order to confirm these results, we have reverse transcribed RNA from pagetic bone and have specifically amplified for distemper and measles sequences using the polymerase chain reaction (PCR) technique. We have found that 8/13 of the patients examined had distemper and 1/10 cases had measles nucleic acids sequestered within their bone cells. One individual was found to have both viruses sequestered in his bone cells. Dideoxy sequencing of the distemper virus PCR products revealed 2% base pair changes in the nucleic acid sequences relative to the Onderstpoort strain of canine distemper. We can conclude that in Pagets disease, canine distemper and possible other paramyxoviruses reside in bone cells and that the persistent nature of the virus may be due to mutations in the viral genome.

Low frequency electric and magnetic fields have different effects on the cell surface

There is a considerable controversy over the nature of weak electromagnetic‐field effects in living organisms. Part of the controversy can be traced to a lack of understanding of whether electric or magnetic fields are involved in producing bioeffects. We find that both 60 Hz electric and magnetic fields alter the cell surface of Physarum polycephalum. Exposure to electric fields increases the negative charge on the cell surface while magnetic‐field exposure decreases the hydrophobic character of the surface. These effects appear to be additive and independent of the waveform of the applied fields.

Expression of a human homeobox-containing gene is regulated by 1,25(OH)2D3 in bone cells

The murine msh-like homeobox-containing gene Msx-2 (Hox-8) has a multiphasic pattern of embryonic expression which includes the developing skull bones and teeth. In order to determine whether this gene might be expressed in adult mineralizing tissues, an adult human bone cell (osteoblastic) cDNA library was screened with a murine Msx-2 probe. One of the positive clones obtained was sequenced in full and shown to be highly homologous to the murine Msx-2 gene. Expression of this gene, MSX2, was studied in human bone-derived cells, where a low level of expression was detected which was greatly induced by stimulation with 1,25(OH)2D3. Little or no increase in expression was detected with retinoic acid or TPA stimulation. In cultures of skin fibroblasts no stimulation of expression with 1,25(OH)2D3 was detected. MSX2 is the first homeobox-containing gene to be shown to be regulated by 1,25(OH)2D3 and to be expressed in adult human bone cells. These results suggest a possible role for MSX2 in bone cell differentiation and/or calcium homeostasis.

Canine distemper virus transcripts detected in the bone cells of dogs with metaphyseal osteopathy

Using the technique of in situ hybridisation, we have recently extended our observations that canine distemper virus (CDV) is present in the bone cells of patients with Pagets disease, and have shown that CDV is also detectable in the bone cells of dogs that are naturally infected with the virus. Since hybridisation was localised to bone cells within the metaphyses of the affected dogs, we investigated the possibility that CDV might be involved in the canine metaphyseal bone disorder, metaphyseal osteopathy. Bone samples from three cases of metaphyseal osteopathy were examined for the presence of the CDV nucleocapsid (CDV-N) gene and the measles virus nucleocapsid (MV-N) gene, using 35S-labelled sense and antisense riboprobes. As with our previous findings in Pagets disease of bone, only the antisense probe was found to hybridize to the osteoblasts and osteoclasts within the affected metaphyses. No hybridisation was seen with the CDV-N sense and MV-N probes in any of the samples tested. Bone samples were also taken from one of the cases to check for the presence of the CDV-N gene using the polymerase chain reaction (PCR). Our findings with in situ hybridisation were confirmed by PCR and subsequent Southern blotting and probing with a 32P-labelled cDNA probe. The detection of CDV RNA within the bone cells of dogs with metaphyseal osteopathy suggests that this virus may be a cause of the disease and provides further, indirect evidence that CDV might be responsible for the bony abnormalities seen in Pagets disease of bone.

The incorporation of glycolipids with defined carbohydrate sequence into liposomes and the effects on partition in aqueous two-phase systems.

A glycolipid with a defined carbohydrate sequence, derived from the glycoprotein fetuin, has been synthesised and incorporated into liposomes. The effect of the glycolipid on partition of the liposomes in aqueous two-phase systems has been investigated. Incorporation of glycolipid into liposomes changed their partition behaviour in a concentration-dependent manner. The effects on partition of the sequential removal of the terminal carbohydrates were investigated. Partition behaviour appeared to be determined by the net effect of a range of factors including the nature of the terminal carbohydrate, interactions of the lipid region of the glycolipid and possibly carbohydrate chain length. The electrostatic potential difference which can be created between the phases (by the inclusion of certain ions, notably phosphate) appeared to have no detectable effect on partition, even when N-acetylneuraminic acid was present as the terminal carbohydrate of the glycolipid.

Cloning and expression analysis of two ZFY-related zinc finger genes from Alligator mississippiensis, a species with temperature-dependent sex determination

In order to investigate the molecular mechanism of temperature-dependent sex determination, a human zinc finger gene (ZFY), known to be highly conserved amongst other species, was used to isolate homologues from the genome of the American alligator, Alligator mississippiensis. ZFY was originally a candidate for the primary testis-determining gene in man, but is now thought to function further down the sex-determining cascade. Two alligator genes are described, Zfc and Znc6. Both code for zinc finger proteins and exhibit amino acid (aa) homologies to ZFY of 91% and 73%, respectively. Znc6 shows aa homology of 88% to the protein encoded by the zinc finger exon of the human ZFY-related gene, ZNF6, recently found on the X chromosome. Analysis of Zfc and Znc6 expression during embryonic development identified two major transcripts of 5.9 kb and 2.7 kb coding for Zfc, whilst only one transcript of 4.8 kb was detected for Znc6. Both genes are transcribed at all stages tested, from day 3 (post egg laying) throughout gestation. The expression level of all transcripts appears to decline towards the time of hatching (65-72 days). No sex-specific differences in the expression were observed. The extensive sequence conservation of the genes between reptiles and humans suggests major functional constraints. The expression patterns indicate that these genes do not play a primary role in temperature-dependent sex determination.

Surface Heterogeneity of Bovine Sperm Revealed by Aqueous Two-phase Partition

Ejaculated, bovine sperm have been subjected to multiple partition in aqueous two-phase systems. This partition, carried out in a countercurrent fashion, reveals heterogeneity of the sperm population with respect to surface properties. The sperm, when partitioned in phase systems that detect non-change associated surface properties (change-insensitive) are largely distributed as two distinct populations. In charge-sensitive phase systems (which principally detect cell surface molecules carrying charge) the sperm do not show any obvious surface heterogeneity. Considerable heterogeneity is revealed in affinity-ligand phase systems containing palmitic acid coupled to one of the phase components-poly(ethylene glycol). There is a difference in surface heterogeneity between sperm which have been washed in buffer or left unwashed, direct from the ejaculate. This is indicative of weak adsorption of proteins to the sperm surface in seminal fluid. These results show that bovine ejaculated sperm is a heterogeneous cell population having unequal distributions of a number of different surface molecules.

An alternatively spliced transcript, p65A2, of the gene encoding the p65 subunit of the transcription factor NF-κB

Abstract We have identified a second alternatively spliced transcript of the NF-KB transcription factor p65 subunit. This transcript, named p65Δ2, varies from p65 at two functionally important regions.

Surface heterogeneity of rat sperm during maturation.

Rat sperm isolated from the caput and caudal epididymis and the vas deferens were subjected to multiple partition in aqueous two-phase systems. The technique was used to reveal heterogeneity of a sperm population with respect to particular surface properties. Sperm from all three regions gave broad distributions indicative of heterogeneous cell populations. Greatest heterogeneity was observed for cauda sperm with caput and was sperm producing similar distributions.Following multiple partition sperm from different regions of the distribution profiles were immunostained with three antibodies known to recognise maturation antigens. The results show that some antigens are acquired during epididymal transit whilst others are present throughout.The partition (surface heterogeneity) seen cannot therefore be explained solely by the distribution of the antigens recognised by 2D6, 6B2 and 3D5.