Genomic hypomethylation in neoplastic cells from dogs with malignant lymphoproliferative disorders.

DNA methylation is an epigenetic modification in which a methyl group is added usually to the fifth carbon position of a cytosine residue. Dysregulation of this process is an important molecular event which has been shown to be associated with neoplastic transformation and tumour progression in humans and mice. Features of methylation dysregulation in many different types of neoplasms include general genomic hypomethylation, focal hypermethylation, and altered expression of genes which encode a series of DNA (cytosine-5) methyltransferases. Interestingly, many types of neoplasia that are recognised in humans also develop spontaneously in the dog. By comparing the restriction patterns of MspI and HpaII, this study demonstrates that as in human, genomic hypomethylation is a feature of neoplastic cells in the majority of canine lymphoma cases and approximately one-third of canine leukemia cases confirming that dysregulation of the DNA methylating machinery is implicated in malignant transformation of lymphoid cells in some dogs.

Novel nucleotide substitutions within the coding region of DNMT2 are in strong linkage disequilibrium in Caucasians and Japanese.

Investigations into mechanims by which cytosine methylation may be genetically controlled have led to the identification of single nucleotide polymorphisms within the coding region of DNMT2 that are conserved in different ethnic groups. The DNMT2 I allele includes a G at nucleotide position 104 of exon 2 and a C at position 50 of exon 4. The alternative allele, DNMT2 II, includes an A and T, respectively, at these positions. G was never found in the absence of C and vice versa and A was never found in the absence of T and vice versa. The gene products of DNMT2 I and DNMT2 II differ by the inclusion of a histidine or tyrosine residue at the position specified by codon 101. This amino acid substitution alters the amino acid composition of a conserved methylating enzyme motif shown to be involved in S-adenosylmethionine binding in M.HhaI, a bacterial methyltransferase that is almost identical to DNMT2 in size and structure. Demonstration of strong linkage disequilibrium between the nucleotide substitutions associated with each DNMT2 allele provides valuable tools for the investigation of molecular genetic mechanisms of evolution and speciation.

Five novel alternatively spliced transcripts of DNA (cytosine-5) methyltransferase 2 in human peripheral blood leukocytes.

Alternative splicing of RNA molecules transcribed from DNA (cytosine-5) methyltransferases has been proposed as a mechanism by which methylation is able to effect diverse biological processes in higher eukaryotes. This study has investigated transcriptional versatility of DNA (cytosine-5) methyltransferase 2, which may methylate cytosine residues within 5'-CCTGG-3' pentanucleotides in regions of the human genome devoid of 5'-CG-3' methylation. Five novel splice variants of DNA (cytosine-5) methyltransferase 2 were identified in the peripheral blood leukocytes of healthy subjects following cloning and sequencing of RT-PCR products amplified using gene specific oligodeoxyribonucleotide primers. The generation of some of these splice variants may be influenced by the formation of secondary structures within pre-mRNA due to the repetition of sequences flanking alternatively spliced exons in a reverse and complementary orientation on the same strand. These findings enable novel approaches to investigate the role of RNA secondary structures in alternative splicing. The DNA (cytosine-5) methyltransferase 2 splice variants are generated in all the major cell types of peripheral blood, as well as in neoplastic lymphoid cells indicating that they are unlikely to generate proteins involved in control of the cell cycle or cellular differentiation. Interestingly, the gene products generated by some splice variants completely or partially lack highly conserved amino acid motifs shown to be important for the catalysis of cytosine methylation. The possibility cannot be excluded, therefore, that alternative splicing of DNA (cytosine-5) methyltransferase 2 pre-mRNA may generate protein isoforms which have different methylating capabilities or which are involved in biological processes other than the catalysis of cytosine methylation.

Safe technique for removal of extensive renal cell tumors.

We describe our technique for excision of a renal tumor that extended to the right atrium and was adherent to the inferior vena cava within the liver. The surgical procedure was performed using cardiopulmonary bypass with deep hypothermic circulatory arrest and retrograde cerebral perfusion to extend the safe period of cerebral ischemia.

The diagnositc significance of Myf-3 hypermethylation in malignant lymphoproliferative disorders.

Deregulated methylation of cytosine in DNA is a frequent finding in malignancy that is reflected by general genomic hypomethylation and regional hypermethylation that includes the myogenic gene Myf-3. In this study of 198 DNA samples from 186 patients with a wide range of lymphoproliferative disorders (LPD), the methylation status of Myf-3 was assessed to evaluate its significance in the diagnosis of malignant LPD. DNA was digested with the restriction endonucleases HpaII and MspI, and using the Southern blot (SB) technique, the size and density of fragments that hybridized with a Myf-3 probe were used to assign the methylation status. None of the samples from 45 patients from a wide age range with benign LPDs had evidence of altered Myf-3 methylation and there was no age-related methylation change. By contrast, 115/123 (93%) of samples from patients with non-Hodgkin lymphoma (NHL) or lymphoid leukemia had increased Myf-3 methylation. There was no methylation alteration in 22/24 (92%) of samples from patients with Hodgkin lymphoma (HL), nor in five of six samples from LPDs that had atypical histopathologic features which were not diagnostic of lymphoma, while the remaining sample of atypical LPD had hypermethylated Myf-3 fragments. There was an association between increasing Myf-3 methylation and higher histopathologic grade of malignancy within specific lymphoma categories. It is concluded that the detection of increased Myf-3 methylation is a sensitive and specific test of malignancy which may complement other molecular methods that are currently used for the assessment of clonality. It may be of particular diagnostic use in natural killer (NK) and null cell malignancies for which other indicators of clonality are lacking. Furthermore, methylation status may prove to be of potential prognostic value.

Fatigue testing median sternotomy closures.

OBJECTIVE: Sternal dehiscence is commonly due to wire cutting through bone. With a biological model, we measured the rate of cutting through bone, of standard steel wire closure, peristernal steel wire, figure-of-eight closure, polyester and sternal bands sternotomy closure techniques. METHODS: Polyester, figure-of-eight, peristernal and sternal band closures were tested against standard closure eight times using adjacent paired samples, to eliminate biological variables. Fatigue testing was performed by a computerized materials-testing machine, cycling between loads of 1 and 10 kg. The displacements at maximum and minimum loads were measured during each cycle. Cutting through, manifested by the displacement at the maximum load between the 1st and 150th cycles was measured. The percentage cut-through of each closure method versus standard closure was calculated. RESULTS: The differences in the displacement between each of the polyester (1.01 mm), figure-of-eight (0.52 mm), peristernal (0.72 mm) and sternal band (0.66 mm) groups versus standard closure (0.22, 0.22, 2.1, 3.2 mm) in the paired samples were statistically significant (Student's paired t-test; P<0.01). There were statistically significant differences in the percentage cut-through of polyester, figure-of-eight, peristernal and sternal bands (ANOVA, P<0.001), versus standard closure. CONCLUSIONS: In our sheep sternum model, we have quantified the differing rate of cutting through bone of five types of median sternotomy closure techniques. We have controlled for bone variables by testing each closure versus standard closure using paired adjacent bone samples. Peristernal and sternal band closure techniques are significantly superior to standard closure. The use of polyester and figure-of-eight closures requires caution.

Evidence that cytosine residues within 5'-CCTGG-3' pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5'-CG-3' dinucleotides.

In contrast to the complex sequence specificities of the prokaryotic DNA methylating systems, the mammalian machinery identified thus far methylates cytosine residues within the context of a 5'-CG-3' dinucleotide. To explore the possibility that cytosine residues that do not precede guanine may be independently methylated in mammalian DNA, we have examined a region of the human myogenic gene, Myf-3, which is not targeted by the methylating system that methylates 5'-CG-3' dinucleotides. Our investigations have revealed cytosine methylation within the 5'-CCTGG-3' pentanucleotides specified by the 0.8-kb Myf-3 probe. We have also found that in DNA from neoplastic cells, in which 5'-CG-3' dinucleotides within Myf-3 become abnormally hypermethylated, cytosine residues within 5'-CCTGG-3' pentanucleotides are not methylated. Moreover, methylation of 5'-CCTGG-3' pentanucleotides was not detected within the closely related Myf-4 gene, which is normally 5'-CG-3' hypermethylated. These findings indicate the existence of a system that methylates 5'-CCTGG-3' pentanucleotides independently of the system that methylates cytosine residues within 5'-CG-3' dinucleotides. It is possible that the 5'-CCTGG-3' methylating system influences the fate of foreign integrated DNA.

Hypomethylation of cytosine 5-methyltransferase in human neoplasms.

Cytosine methylation is an epigenetic modification of DNA involved in control of gene expression. Neoplastic cells exhibit various alterations both in DNA methylation and activity of the enzyme responsible for this modification, 5-methyltransferase (5-MeTase). As there is little requirement for 5-methyltransferase expression in normal cells except during mitosis, we argued that the gene would be hypermethylated in normal cells. Southern analysis revealed almost complete methylation of the gene in genomic DNA from the peripheral blood leukocytes of healthy subjects and a primary fibroblast derived cell line. In contrast, in DNA from a range of tumour tissues and tumour derived cell lines, 5-MeTase exhibited marked hypomethylation. The results of this study indicate that dysregulation of the DNA methylating machinery, especially with respect to the methylation status of 5-MeTase, is a feature of a wide range of neoplasms.

Allele-specific variation in the gene copy number of human cytosine 5-methyltransferase.

Previously, we have identified two alternate allelic forms of cytosine 5-methyltransferase, 5-MT I and 5-MT II, specified by polymorphic fragments of 1.5 and 1.1 kb, respectively. In the presence study, a 0.8-kb genomic probe was prepared which was confirmed to be included within the polymorphic fragments. The 0. 8-kb probe hybridised with greater intensity to the 1.1-kb fragment than the 1.5-kb fragment. Densitometric analysis indicated that there is 1 copy of 5-MT associated with 5-MT I, whereas there may be 1-4 copies of the gene associated with the 5-MT II allele. Segregation studies demonstrated that the multiple copies of 5-MT II are inherited in a Mendelian fashion. These results allow novel approaches to investigating the underlying mechanisms of cytosine methylation and gene duplication.

Characterization of the alternate allelic forms of human PAX7.

Six different allelic forms of the human neurogenic and myogenic developmental gene, PAX7, have been identified. They are distinguished by the number of tandem tetranucleotide, GAAG, repeats at a polymorphic site within the second intron of the paired box. Within the same intron, a second polymorphic site was found to have variable numbers of a dinucleotide TG repeat. The alleles are identified by a PCR-based method with oligo primers that span the variable regions of the intron. Several of the alleles include a duplicate copy of the entire paired box. Segregation studies demonstrate that the PAX7 alleles are inherited in a Mendelian fashion and that the duplicate copies of the PAX7 paired box region present in some of the alleles are closely linked. This initial study identified differences in the distribution of PAX7 alleles in DNA from patients with the skeletal muscle myopathy, dermatomyositis. Recognition of genetic polymorphism of PAX7 allows new approaches to understanding the role of PAX7 in myogenesis, neurogenesis, and neuromuscular disorders.

Routine sternal closure using interlocking multitwisted wires.

We describe a method of sternal closure that enhances sternal stabilization and minimizes bleeding from sternal fractures caused by retraction. With the technique of interlocking multitwisted wires the initial placement of the wire sutures is the same as in traditional sternal closure, however the twisting technique is improved, with multiple twisting including four twisted strands. Our method of closure is effective, simple and quick to perform and has several advantages over conventional or figure-of-eight closure. This closure is also biomechanically more rigid than conventional or figure-of-eight closure. We therefore recommend routine sternal closure using interlocking multitwisted wires.

Pax7 is expressed in the capsules surrounding adult mouse neuromuscular spindles.

The multigene Pax family of transcription factors plays an important role in the development of the central nervous system as well as in organ morphogenesis. Expression of one of the members of the family, Pax7, has been described in embryonic muscle and in both embryonic and adult brain. We recently detected Pax7 transcripts in RNA isolated from adult mouse skeletal muscle and brain and here use in situ hybridisation to localise the expression within these tissues. Pax7 expression was observed in neural cells of the brain and in cells of neural crest origin in the inner and outer capsules of neuromuscular spindles. The results suggest that Pax7 may be implicated in the formation and maintenance of neuromuscular contacts within the muscle spindle throughout life.

A biomechanical study of median sternotomy closure techniques.

OBJECTIVE: Sternal dehiscence is a complication of median sternotomy incisions with high mortality and morbidity. Different techniques of sternal closure have been described. Rigid fixation of the sternum results in earlier union. We measured the rigidity of sternotomy fixation using a mechanical model in order to differentiate different techniques of sternal closure using their biomechanical characteristics. METHODS: We measured the force-displacement curves of six different fixation techniques using a metal sternal model using a computerized materials-testing machine. We tested straight wires (the most commonly used surgical technique), figure-of-8 wires, 'repair' technique (used when a wire breaks), Ethibond, Sterna-band and a 'multitwist' closure described for the first time. RESULTS: At 20 kg force, twisted wires used for sternotomy closures start to untwist. The most rigid closure was a multitwist closure that displaced only 0.37 mm at a force of 20 kg. Straight wires displaced 0.78 mm, figure-of-8 wires 1.20 mm, Sterna-band 1.37 mm, repair wires 5.08 mm, Ethibond 9.37 mm. The single factor Anova test for the rigidity of the different closures had P-values <0.0001. CONCLUSIONS: We applied a mathematical model to calculate chest wall forces during coughing, in order to determine the force placed upon a sternotomy closure. We conclude that severe coughing may cause wires to untwist. We discuss potential applications of different wire closures based on their characteristics.

Alternate Pax7 paired box transcripts which include a trinucleotide or a hexanucleotide are generated by use of alternate 3' intronic splice sites which are not utilized in the ancestral homologue.

In the mouse, Pax7 plays an important role in development of the skeletal muscles of the limbs, elements of the nervous system and cranio-facial structures. It is expressed in the brain and skeletal muscles of the limbs in the mature mouse. Recently, we have identified alternate transcripts that differ by inclusion or exclusion of a trinucleotide and/or a hexanucleotide in the paired domain encoding region. Sequencing of the paired box in genomic DNA from SJL/J and BALB/c mice reveals that the trinucleotide and hexanucleotide are generated by selection of alternate splice sites at the 3' terminus of each of the two paired box introns, respectively. The proximal 3' splice site of the first intron, which includes the trinucleotide in the mature transcript, is preferentially selected in skeletal muscle and brain. By contrast, the proximal 3' splice site of the second intron, which results in inclusion of the hexanucleotide in the mature transcript, is preferentially selected only in skeletal muscle. The distal alternate 3' splice site, which results in exclusion of the hexanucleotide in the mature transcript, is preferentially selected in the brain. These findings raise the possibility that there may be tissue-specific factors that influence the specificity of the spliceosomal machinary. Reference to the structure of the proposed primordial form of Pax7 suggests that the ability to utilize the alternate splice sites that generate inclusion of the trinucleotide and the hexanucleotide in the mature transcripts may have occurred in recent evolutionary times.

Haemolysis during cardiopulmonary bypass: an in vivo comparison of standard roller pumps, nonocclusive roller pumps and centrifugal pumps.

Cardiopulmonary bypass (CPB) involves the use of either an occlusive roller pump or centrifugal pump. Damage to blood elements, including haemolysis, may arise from occlusion when using a roller pump; the appropriate degree of occlusion has not yet been determined scientifically. Centrifugal and nonocclusive roller pumps are reputed to reduce haemolysis. The objective of this study was to compare haemolysis caused by a standard roller pump with a dynamically set nonocclusive roller pump and with a centrifugal pump. We prospectively randomized 60 patients undergoing routine coronary artery surgery into three groups: standard roller pump (STD, n = 20), dynamically set roller pump (DYN, n = 20), or centrifugal pump (CEN, n = 20). The level of plasma free haemoglobin (FHb) was measured preoperatively, and the rate of formation of FHb (in mg/dl/min) was determined at the end of the ischaemic phase and at the end of CPB. Cardiotomy suction blood was isolated for the ischaemic phase and returned before the end of CPB. It was found that there were no differences between the groups in demographic or operative variables. The rate of formation of FHb at the end of the ischaemic phase was similar for all groups (STD 0.108 +/- 0.10, DYN 0.117 +/- 0.08, CEN 0.129 +/- 0.07). At the end of CPB, after return of the cardiotomy suction blood, there was a significant (< 0.001) increase in the rate of formation of FHb in all groups. The increase was similar for each of the groups (STD 0.424 +/- 0.17, DYN 0.481 +/- 0.20, CEN 0.471 +/- 0.18). We conclude that the rates of haemolysis are similar for each of the pump types, and no benefit is conferred by the use of either a dynamically set roller pump or a centrifugal pump compared with the standard roller pump. The return of the cardiotomy suction blood to the circulation is the principal source of plasma free haemoglobin.

Enhancing liver blood flow after cardiopulmonary bypass: the effects of dopamine and dopexamine.

Liver blood flow is reduced after cardiopulmonary bypass (CPB) and both dopamine and dopexamine are used to overcome this. This study compares the effects of these agents on liver blood flow. Thirty patients undergoing elective coronary artery bypass graft surgery were randomized into three groups (n = 10 per group). Six hours after surgery baseline liver blood flow was determined by the percentage disappearance rate of indocyanine green measured by dichromatic auricular densitometery. Patients then received infusions of either: (1) placebo (dextrose 5%); (2) dopamine (4 micrograms/kg/min); (3) dopexamine (1 microgram/kg/min increasing to 2 micrograms/kg/min). One hour after infusion, liver blood flow measurements were repeated. In the dopexamine group the infusion was increased and the measurements repeated another hour later. We found that patient-specific variables and operative details were similar for all groups. Postoperative cardiac index and heart rate were increased significantly by dopamine (cardiac index 2.82 +/- 0.46 l/m/m2 vs 3.28 +/- 0.67 l/m/m2: p < 0.001 and heart rate 87.5 +/- 13.2 vs 96 +/- 16: p < 0.05) and dopexamine at 2 micrograms/kg/min (cardiac index 2.71 +/- 0.53 l/m/m2 vs 3.45 +/- 0.67 l/m/m2: p < 0.05 and heart rate 89.0 +/- 18.9 vs 107.4 +/- 13.6: p < 0.001) compared to placebo (cardiac index 2.97 +/- 0.8 l/m/m2 vs 3.18 +/- 0.9 l/m/m2: p > 0.05 and heart rate 77.2 +/- 7.4 vs 77.3 +/- 8: p > 0.05) despite similar atrial and systemic arterial pressures. The disappearance rate of indocyanine green was not altered during infusion of placebo group (9.0 +/- 3.2%/min vs 7.9 +/- 3.0%/min: p > 0.05) or dopexamine at 1 microgram/kg/min (9.7 +/- 3.1%/min vs 11.2 +/- 4.1%/min: p > 0.05). The disappearance rate was increased with dopamine (6.7 +/- 3.7%/min vs 11.8 +/- 3.0%/min: p < 0.05) and dopexamine 2 micrograms/kg/min (9.7 +/- 3.1%/min vs 13.5 +/- 3.2%/min: p < 0.05). This indicates a 76% increase in liver blood flow with dopamine and a 38% increase with dopexamine. We conclude that dopamine 4 micrograms/kg/min and dopexamine 2 micrograms/kg/min increase liver blood flow, although this may, in part, be related to an increase in cardiac output. Dopexamine shows no advantage over dopamine in enhancing liver blood flow after CPB.

A conserved TN8TCCT motif in the octapeptide-encoding region of Pax genes which has the potential to direct cytosine methylation.

Our previous findings have shown that the developmental genes Pax7 and Pax3 are differentially methylated; the gene region that encodes the paired domain is hypomethylated, whereas the region that encodes the homeodomain is hypermethylated. For this reason, the known DNA sequence between the paired and homeoboxes was analysed for the presence of a conserved DNA motif to which a modifying protein could bind in order to direct the methylation or demethylation of surrounding gene sequences. The octapeptide-encoding region was found to contain several nucleotides that were highly conserved throughout the Pax gene family from phylogenetically distant species. The most conserved nucleotides are thought to comprise a motif TN8TCCT where N8=any combination of eight nucleotides. A conserved octapeptide-like-encoding sequence containing the TN8TCCT motif was also found in non-Pax genes of higher eukaryotes and in the non-coding strand of plants. Moreover, differential methylation seems to be associated with the presence of the TN8TCCT motif in p53 and the human oestrogen receptor genes. The presence of the TN8TCCT motif within an octapeptide-like-encoding sequence in human T-cell leukaemia virus type 1 might suggest that the putative recognition motif may have been introduced into various host genomes via some form of retroviral agent.

Differential expression of four alternate Pax7 paired box transcripts is influenced by organ- and strain-specific factors in adult mice.

The developmental paired-type gene Pax7 is expressed in skeletal muscle and brain during development and in the adult mouse. In this study, RNA was isolated from the brains and skeletal muscles of the limbs of adult BALB/c and SJL/J mice to determine whether there were alternate transcripts which could account for the biological diversity of Pax7. Four alternate transcripts have been identified, each of which differs by the presence and/or absence of a trinucleotide or a hexanucleotide in the region of Pax7 which encodes the paired domain. Inclusion of the trinucleotide and the hexanucleotide results in insertion of the amino acids glutamine (Q) and glycine (GL), respectively, into the paired domain. The insertion of GL is predicted to influence the binding specificity, whereas insertion of Q may affect the relative binding affinity of the N and C termini of the paired domain. The transcripts which include the hexanucleotide are more frequent in RNA from the hind-limb skeletal muscles of adult mice compared with the brain, suggesting that they may effect some form of myogenic function. By contrast, it is possible that transcripts which lack the hexanucleotide encode factors which are involved in neurogenesis. The proportion of the potential myogenic transcript Pax7b was found to be increased in RNA from limb skeletal muscles of adult SJL/J mice compared with that of BALB/c mice. These results provide a new basis for examination of the genetic control of skeletal-muscle formation and neurogenesis.

A novel MspI PCR-RFLP in the human cytosine 5-methyltransferase gene: lack of relevance for malignant lymphoproliferative disease and breast cancer.

Two alternate allelic forms of human cytosine 5-methyltransferase, 5-MT I and 5-MT II, which differ by the absence or presence of an intronic MspI recognition sequence, have been recognised. The polymorphic region was localised using a series of subprobes prepared upon MspI digestion of the 2.5-kb cDNA probe (hmt-2.5). A PCR-based method was then developed for rapid 5-MT genotyping. The gene and phenotype frequencies of 5-MT I and 5-MT II were not significantly different in genomic DNA samples from a series of non-Hodgkin's lymphomas and breast cancer cases compared with DNA from normal subjects. Allelism of 5-MT allows new approaches to the assessment of variation in gene copy number of 5-MT in different types of neoplasia.