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Biomedical subjects

P H Kay

Publications and source records attributed to P H Kay.

At least 37 records · Page 2Linked to original sources
PubMed1998

Pax7 includes two polymorphic homeoboxes which contain rearrangements associated with differences in the ability to regenerate damaged skeletal muscle in adult mice.

Pax7 is a paired-type homeobox gene which has previously been shown to play an important role in skeletal muscle formation. It is expressed in skeletal muscle of the limbs during embryogenesis and in adulthood. The aims of this study were firstly to determine the degree of polymorphism of Pax7 amongst inbred laboratory mice using Southern blotting and Pax7 regional specific sub-probes. Secondly, functional studies were performed on mice with each of the different structural forms of Pax7 to determine whether they were associated with differences in the ability to regenerate damaged skeletal muscle. Four different allelic forms of Pax7 have now been identified in laboratory mice indicating that the previously reported DNA sequence of Pax7 is not applicable to all laboratory mice. Hybridisation patterns of TaqI digested DNA representing each of the different Pax7 alleles with the Pax7 specific sub-probes suggested that in contrast to previous findings, Pax7 is associated with two highly polymorphic homeoboxes. The presence of two homeoboxes in BALB/c mice has been confirmed by DNA sequencing. Results of functional studies have also shown that the ability to regenerate damaged skeletal muscle in adult mice is strongly associated with the presence of a 0.15-kb TaqI fragment derived from one of the homeoboxes.

Alleles↗
PubMed1997

Variation in the methylation profile and structure of Pax3 and Pax7 among different mouse strains and during expression.

Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.

Animals↗
PubMed1997

Haemodynamic performance of a 16-mm Carbomedics aortic prosthesis.

A 55-year-old lady underwent repeat aortic valve replacement using a 16-mm Carbomedics prosthesis. She made an uneventful postoperative recovery and now leads an unrestricted life. Doppler echocardiography reveals a 21-mm Hg gradient across the valve at rest. This did not increase with an infusion of 30 mcg/kg per min of dobutamine, which resulted in an increase in the cardiac output from 1.96 to 5.46 l/min.

Aortic Valve↗
PubMed1997

Alternate Pax7 transcripts are expressed specifically in skeletal muscle, brain and other organs of adult mice.

Pax7 is associated with formation of skeletal muscle and the neural tube in developing embryos. Interestingly, in adult mice, rearrangements of Pax7 are associated with differences in the efficiency of skeletal muscle regrowth between mouse strains. The aim of this study was to investigate the possibility that Pax7 is expressed in skeletal muscle or other tissues from adult mice. Total RNA was isolated from adult mouse tissues and the polymerase chain reaction was performed on reverse transcribed mRNA using primers specific for regions that encode the paired and homeodomain of Pax7. At least four different Pax7 transcripts were found. A full-length transcript similar in sequence to that published previously was identified in skeletal muscle, brain and spleen cells of adult mice. Further putative full-length Pax7 transcripts, including one that contains a hexanucleotide insertion in the paired box and one in which approximately 10 bp have been deleted in the homeobox, were found to be expressed in skeletal muscle and brain of adult mice, respectively. A truncated Pax7 splice product comprising the paired box only was found to be expressed in most adult tissues except liver. Results of these studies demonstrate that there are alternate transcripts of Pax7, some of which are expressed exclusively in adult skeletal muscle and brain. It is possible that one of these transcripts may specify an alternate myogenic pathway involved in regeneration of damaged skeletal muscle in adult mice.

Alternative Splicing↗
PubMed1997

Investigation of the influence of the Mm musculus MyoD1 allele on the efficiency of skeletal muscle regeneration.

MyoD1 alleles distinguish between the Mm musculus and Mm domesticus subspecies of Mus musculus. SJL/J mice, which belong to the Mm musculus subspecies, are able to regenerate skeletal muscle more efficiently than BALB/c mice which possess the Mm domesticus MyoD1 allele. Hence the possibility that regeneration responses are due to species-specific genetic differences including MyoD1 was investigated in this study. Quantitative morphometric analysis following muscle crush injury of 2 Mm musculus strains, MBK and MGL, has indicated that regeneration phenotype is not species-specific and may not be directly related to MyoD1 alleles. These results contrast with prior investigations conducted in SJL/J and BALB/c mice, and indicate that other genes may have a direct influence on skeletal muscle regeneration.

Alleles↗
PubMed1997

B-cell target DNA quantity is a critical factor in the interpretation of B-cell clonality by PCR.

Criteria for the assessment of clonality by Southern blotting are well established but this is not the case for polymerase chain reaction (PCR)-based assays. Our studies, and infrequent reports in the literature, indicate that B-cell clonality may be erroneously inferred if only small numbers of polyclonal B-cells are present in test samples. In order to establish criteria to minimise the false positive assignment of B-cell clonality, DNA was analysed in a semi-nested PCR to detect rearrangement of the immunoglobulin heavy chain gene using a range (1 microgram-0.1 ng) of target DNA amounts from four tonsils and five lymph nodes showing reactive follicular hyperplasia, and from six B-cell lymphomas. A discrete, narrow band of PCR product of constant size was detected throughout the range of target DNA amounts in most lymphomas indicating the presence of a monoclonal B-cell population. In contrast, from the non-malignant tonsils and lymph nodes, larger target amounts generated a broad band of PCR products indicating populations of polyclonal B-cells, but smaller target amounts generated discrete, narrow PCR product bands of inconstant size indicating oligo- or monoclonal B-cell populations. Results of this study demonstrate that a range of DNA target amounts should be tested when the proportion of B-cells in a sample is unknown, thus preventing the analysis of insufficient target DNA which may lead to the false assignment of clonality.

Clone Cells↗
PubMed1997

DNA methylation and developmental genes in lymphomagenesis--more questions than answers?

There is now considerable evidence suggesting that alterations in the DNA methylating machinery play an important role in tumorigenesis and tumour progression. For example, focal hypermethylation and generalised genomic demethylation are features of many different types of neoplasms. It is thought that tumorigenesis and tumour progression may be caused by hypermethylation induced mutational events and silencing of genes which control cellular proliferation and/or demethylation induced reactivation of genes which may only be required during embryological development. Consequently, we have begun to investigate the role of DNA methylation and developmental genes in malignant lymphoproliferative diseases. Previously, in all cases of non-Hodgkins lymphoma and leukemia studied, we have shown that the myogenic developmental gene Myf-3 is abnormally hypermethylated. In this review we discuss the possible significance of these findings since in vitro studies suggest that Myf-3 may play an important role in control of the cell cycle and therefore lymphomagenesis. In vitro and in vivo evidence suggests that PAX genes may also have oncogenic potential. The PAX family of developmental genes are involved in cellular differentiation, proliferation and cell migration. Expression of PAX3 in particular is associated with cellular mobility. Our previous studies have indicated that alternate regional expression of PAX genes may be controlled by DNA methylation. Therefore, we have proposed that abnormal methylation profiles of PAX3 may be associated with neoplastic transformation and/or metastatic potential. Results thus far reveal that the paired box of PAX3 is abnormally hypermethylated and the homeobox abnormally hypomethylated in lymphomas and leukemias. These new findings are consistent with our postulate and support the idea that inappropriate methylation induced activation or inactivation of developmental genes such as Myf-3 and PAX3 play an important role in lymphomagenesis and disease progression and that inspection of the methylation status of other developmental genes is warranted.

Animals↗
PubMed1996

Polymorphism of the mouse E2A gene due to an intronic deletion of 536 bp.

Examination of genetic polymorphism of the transcription factor-encoding gene E2A in laboratory and wild mice by Southern blotting has revealed the presence of two alleles. The most frequent allele is found in Mus musculus (Mm) musculus, as well as Mm domesticus. The less common allele is restricted to the Mm domesticus subspecies. Characterisation of these alleles has shown that the less common allele contains a deletion of approx. 500 bp located within a 1.8-kb intron immediately upstream from the E12 basic helix-loop-helix exon. DNA sequencing determined the deletion to span 536 bp including nucleotides 1045-1580 of the intron within the common allele. The deleted region includes several sequences with similarity to gene regulatory motifs; however, expression of E12 and intron splicing appeat unaltered. The occurrence of an identical deletion in mice from different geographical regions suggests that the uncommon allele may have a long evolutionary history.

Alleles↗
PubMed1995

Association of an unusual form of a Pax7-like gene with increased efficiency of skeletal muscle regeneration.

Efficiency of regeneration of mechanically injured skeletal muscle is more pronounced in SJL/J mice, as compared to other laboratory strains in which regenerative properties of skeletal muscle are uniformly poor. Previously, we postulated that a small number of genes might differ between SJL/J and other mouse strains, and would be responsible for this variation in the efficiency of skeletal muscle regeneration. The results of initial experiments demonstrated that SJL/J mice have a unique form of the myogenic gene, Myo-D1, which partly influences efficiency of skeletal muscle repair, and that other genes were also involved. To identify other candidate genes, differences were sought within the myogenic paired box/homeobox-containing gene Pax7 between SJL/J and other laboratory mouse strains. Southern blotting indicated that SJL/J, Quackenbush and DDO mice share a Pax7/TaqI RFLP which differs from all other laboratory strains tested. This RFLP is most likely due to sequence differences within the homeobox of a Pax7-like gene. In vivo studies revealed that Quackenbush and DDO mice also share the same regenerative properties of mechanically damaged skeletal muscle as SJL/J mice. Since Quackenbush and DDO mice lack the SJL/J type of Myo-D1, and DDO belong to a different mouse sub-species, these studies suggest that structural alterations in the homeobox of a Pax7-like gene may be implicated in the effectiveness of renewal of damaged skeletal muscle of the limb in the mature animal.

Animals↗
PubMed1995

Polymorphism of the murine inhibitor of differentiation-controlling gene, Id.

The structure of Id was examined by Southern analysis in inbred mouse lines which included five subspecies of Mus musculus. No variation was detected within this species. The species Mus cookii shares the same form found in mice of M. musculus derivation, indicative of a long evolutionary history and a common origin. However, five TaqI polymorphisms were found among several Mus species, suggesting that Id has been modified throughout species diversification. Whether these variants impart functional changes is yet to be determined.

Animals↗
PubMed1995

Studies on the polymorphism of the fifth component of complement in laboratory mice.

The fifth component of complement C5, as well as C3, plays an important role in the inflammatory response. Since our previous studies have implicated polymorphism of C3 in disease pathogenesis, similar studies were undertaken on C5 using inbred and outbred laboratory mice. Consistent with previous findings, male mice were shown to have higher serum C5 concentrations than female mice. In male SJL/J mice, however, the serum concentration of C5 was significantly greater than in males of other strains. An immunofixation electrophoretic method was therefore developed to determine whether SJL/J mice had a different electrophoretic form of C5. The electrophoretic mobility of C5 in serum from SJL/J mice was similar to that in other strains. Some mice, including NOD/Lt, Quackenbush and ARC, had complete C5 deficiency. Southern blotting showed that C5 deficiency in these mice was associated with the same gene rearrangement found in other C5-deficient laboratory mice. No difference in the structure of the C5 gene was seen between SJL/J mice and other C5-sufficient inbred strains. In contrast, C5-sufficient Quackenbush and ARC outbred mice had a unique C5 RFLP pattern.

Animals↗
PubMed1994

Evidence for adenine methylation within the mouse myogenic gene Myo-D1.

Previous studies have indicated that there may be uncleavable TaqI sites (TCGA) within the mouse myogenic gene, Myo-D1. Fragments of DNA bearing most of the presumed insensitive TaqI sites have been reproduced using PCR. The presence of each of the originally uncleavable TaqI sites has been confirmed and each TaqI site has been shown to be sensitive to TaqI hydrolysis in PCR-synthesized genomic DNA. Since TaqI is inhibited by methylation of the adenine residue within its recognition sequence (but not by cytosine methylation), it is suggested that specific adenine bases are methylated in the coding region of Myo-DI and maintained throughout cell division. The same TaqI recognition sequences are insensitive to digestion in genomic DNA isolated from various mouse tissues including fetus, regenerating skeletal muscle and a myogenic cell line, all of which express Myo-D1. Thus, adenine methylation is not a modification of DNA following gametic fusion nor does it appear to play a major role in regulation of Myo-D1 expression.

Adenine↗
PubMed1994

Polymorphism of the mouse transcription factor-encoding gene E2A.

Southern blotting analysis using a cDNA probe consisting of the central portion of the E12 coding region has revealed two distinct forms of E2A, one which is common to all inbred and wild mouse strains derived from Mus musculus musculus and Mus musculus domesticus, whereas the other is less common and has only been found in the wild mouse population of Mus musculus domesticus.

Animals↗