The effects of multidisciplinary rehabilitation in patients with early-to-middle-stage Huntington's disease: a pilot study.

BACKGROUND AND PURPOSE: Despite advances in the understanding of Huntington's disease (HD), treatment remains symptomatic. Multidisciplinary rehabilitation, however, appears to impact disease progression. Here we show the feasibility, safety and efficacy of a 9-month multidisciplinary rehabilitation programme in a small cohort of patients with early-to-middle-stage HD. METHODS: Twenty patients with HD were assigned to two groups, equally matched for cognitive and motor scores. One group received the intervention, whilst the other served as control. The Unified-Huntington's-Disease-Rating-Scale-Total-Motor-Score was the primary outcome measure. Neurocognitive/psychological tests, body composition, postural stability, strength and quality of life assessments were secondary outcome measures. RESULTS: The intervention reduced motor and postural stability deterioration, with minor improvements in depression, cognition and quality of life. Significant gains were observed for fat-free mass and strength. CONCLUSION: This pilot study suggests that a prolonged multidisciplinary rehabilitation programme in early-to-middle-stage HD is feasible, well-tolerated and associated with therapeutic benefit. Further explorative, larger studies are warranted.

Changing Pax6 expression correlates with axon outgrowth and restoration of topography during optic nerve regeneration.

Pax6, a member of the highly conserved developmental Pax gene family, plays a crucial role in early eye development and continues to be expressed in adult retinal ganglion cells (RGCs). Here we have used Western blots and immunohistochemistry to investigate the expression of Pax6 in the formation and refinement of topographic projections during optic nerve regeneration in zebrafish and lizard. In zebrafish with natural (12-h light/dark cycle) illumination, Pax6 expression in RGCs was decreased during axon outgrowth and increased during the restoration of the retinotectal map. Rearing fish in stroboscopic illumination to prevent retinotopic refinement resulted in a prolonged decrease in Pax6 levels; return to natural light conditions resulted in map refinement and restoration of normal Pax6 levels. In lizard, RGC axons spontaneously regenerate but remain in a persistent state of regrowth and do not restore topography; visual training during regeneration, however, allows a stabilization of connections and return of topography. Pax6 was persistently decreased in untrained animals but remained increased in trained ones. In both species, changes in expression were not due to cell division or cell death. The results suggest that decreased Pax6 expression is permissive for axon regeneration and extensive searching, while higher levels of Pax6 are associated with restoration of topography.

Autoantibody Production in Cancer--The Humoral Immune Response toward Autologous Antigens in Cancer Patients.

A link between autoimmune responses and cancer via autoantibodies was first described in the 1950s. Since, autoantibodies have been studied for their potential use as cancer biomarkers, however the exact causes of their production remain to be elucidated. This review summarizes current theories of the causes of autoantibody production in cancer, namely: 1) defects in tolerance and inflammation, 2) changes in protein expression levels, 3) altered protein structure, and 4) cellular death mechanisms. We also highlight the need for further research into this field to improve our understanding of autoantibodies as biomarkers for cancer development and progression.

Delta 6-desaturase: improved methodology and analysis of the kinetics in a multi-enzyme system.

A new method of assay for the delta 6-desaturation of linoleic acid was developed. This method, which uses HPLC for separation of the fatty acid substrate and product, exhibited a lower coefficient of variation (0.3%) than the reported TLC method (3.5%), and avoided the step of methylation of the saponified fatty acid substrate and product. Using this new method of assay, the kinetics of the delta 6-desaturase in a multi-enzyme system were analysed. A number of factors that could have striking effects on desaturase kinetics were investigated, including the effect of (i) endogenous microsomal linoleic acid on total substrate concentration, and (ii) the pre-reaction catalysed by acyl-CoA synthetase and competing reactions catalysed by lysophospholipid acyltransferase and acyl-CoA hydrolase. Endogenous free linoleate in the hepatic microsomes was found to be 2.9 +/- 1.0 microM (0.5 mg microsomal protein/ml), which was comparable to added substrate concentrations (1.8 to 7.9 microM). The kinetics of the delta 6-desaturase were dissected from the kinetics of the above mentioned pre-reaction and competing reactions through a combination of experimental approaches and computer modeling. From computer modeling, a Km and Vmax of 1.5 microM and 0.63 nmol/min were calculated for the delta 6-desaturase, compared to Km and Vmax of 10.7 microM and 0.08 nmol/min calculated directly from data uncorrected for endogenous substrate. It was concluded that lysophospholipid acyltransferase, acyl-CoA synthetase and endogenous linoleic acid significantly affect the kinetic measurements of hepatic microsomal delta 6-desaturase. These results have implications for kinetic analyses of all desaturates in microsomal systems.

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.

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 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.

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.

Aberrant PAX3 and PAX7 expression. A link to the metastatic potential of embryonal rhabdomyosarcoma and cutaneous malignant melanoma?

Transcription factors encoded by PAX3 and PAX7 are amongst the first expressed in the embryo, being principal regulators of neurogenic and myogenic progenitor cell specification and embryonic segmentation. The basis for this review lies in the supposition that genetic programs for cell migration, thought regulated by PAX3 and PAX7 during embryonic development, become tools used by the metastatic cell. In highly metastatic neoplasms arising from cells of neurogenic and myogenic lineages such as embryonal rhabdomyosarcoma and cutaneous malignant melanoma, markedly high expression levels of PAX3 and PAX7 support this supposition. As PAX3 and PAX7 are known to play a role in the regulation of migratory events in embryogenesis, it is possible that the metastatic potential of these tumours is directly linked to migratory properties conferred them through PAX expression. Here we provide a novel perspective by correlating metastasis with expression of PAX3, PAX7 and ephrin/Eph receptors as well as NCAMs, cell surface markers normally involved in migration and adhesion during development, and propose a role for PAX genes in the increased metastatic potential of these tumours.

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration.

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.

The relation between the PST1 restriction fragment length polymorphism at the APO-AI locus and plasma APO-AI concentrations in marathon runners.

A PST1 restriction fragment length polymorphism (RFLP), located close to the apolipoprotein AI (apo-AI) gene on chromosome 11, is associated with elevated levels of apo-AI in normal healthy individuals and with depressed levels in patients with coronary heart disease. In both cases the association is with the P2 allele (the allele not containing the PST1 cutting site). Prolonged exercise is known to increase steady-state plasma apo-AI concentrations. We investigated the effect of adaptation to endurance exercise on the association of the PST1 marker with plasma apo-AI levels. Eighty-two male subjects between the ages of 20 and 50 years were randomly selected from a group of local marathon runners. The frequency of the P2 allele was 14% in this group. This was similar to the frequency reported for this RFLP in other population groups of healthy men. Plasma levels of apo-AI were elevated in the marathon runners compared with randomly selected healthy South African men in the same age group. There was, however, no association between the PST1 RFLP and the plasma high-density lipoprotein cholesterol and/or apo-AI concentrations in this group. The elevated apo-AI levels in marathon runners therefore bear no relation to differences associated with the PST1 RFLP at the apo-AI gene locus.

The effect of fluroxene [(2,2,2-trifluoroethoxy)ethane] on haem biosynthesis and degradation.

Acute fluroxene treatment of male Wistar rats decreases the amounts of hepatic microsomal cytochrome P-450 and haem, increases the activities of hepatic delta-aminolaevulinate synthase and haem oxygenase, and increases the amounts of haem precursors (delta-aminolaevulinate and porphobilinogen) in the urine. All of the above effects of fluroxene are enhanced by pretreatment of the experimental animals with 3-methylcholanthrene and phenobarbital. The amounts of porphyrins in the urine and faeces were generally unaffected by acute fluroxene treatment of uninduced or 3-methylcholanthrene- or phenobarbital-induced Wistar rats. 2,2,2-Trifluoroethyl ethyl ether, the saturated analogue of fluroxene, did not affect the amounts of hepatic cytochrome P-450 and haem, the amounts of any of the haem precursors in the urine or faeces, or the activity of hepatic haem oxygenase in phenobarbital-induced male Wistar rats. The amounts of hepatic cytochrome P-450 and haem and of the haem precursors in urine and faeces, and the activity of delta-aminolaevulinate synthase, were generally not altered by acute fluroxene treatment of uninduced male Long-Evans rats. Chronic treatment of Wistar rats with fluroxene resulted in small increases in the amounts of delta-aminolaevulinate and porphyrins in urine. The amounts of porphobilinogen in urine were elevated up to 2000%, whereas the amounts of the porphyrins in faeces were generally unaffected. After chronic fluroxene treatment, the activity of delta-aminolaevulinate synthase was increased, whereas the activity of uroporphyrinogen synthase was decreased. It is concluded that acute fluroxene treatment may affect haem biosynthesis and degradation by a mechanism similar to allylisopropylacetamide, namely by stimulating an atypical cytochrome P-450-dependent pathway for haem degradation. The effects of chronic fluroxene treatment on haem biosynthesis may be a consequence of this mechanism or a result of the inhibition by fluroxene of uroporphyrinogen synthase. Chronic fluroxene treatment of male rats affects the haem biosynthetic pathway in a manner similar to that seen in human genetic acute intermittent porphyria.

1,1,1-Trichloropropene-2,3-oxide: an alternate mechanism for its inhibition of cytochrome P-450.

Incubation of hepatic microsomes from phenobarbital treated rats, 1,1,1-trichloropropene-2,3-oxide (TCPO) (3 mM), EDTA (0.2 mM) and NADPH-gene-rating system in vitro decreased the levels of cytochrome P-450 by 40% and equivalently decreased microsomal heme. Appreciable losses of cytochrome P-450 were not observed [1] if metyrapone (2.3 mM) or CO:O2 (80:20; v/v) were included in incubation mixtures, [2] if the TCPO or NADPH-gene-rating system were omitted from reaction mixtures, or [3] if microsomes from untreated or beta-naphthoflavone treated rats were utilized. The time course for the TCPO mediated loss of hepatic microsomal cytochrome P-450 showed a time lag of 5 min, before the levels of cytochrome P-450 were significantly altered, followed by a 10-15 min period in which the levels of cytochrome P-450 rapidly decreased to a non-zero plateau level. The concentration of TCPO required for half maximal loss of cytochrome P-450 was ca. 0.5 mM. In the absence of cytochrome P-450 degradation, TCPO (2 mM) was an effective inhibitor of aminopyrine demethylase and benzpyrene-3-hydroxylase but not of p-nitroanisole demethylase or ethoxyresorufin deethylase. In contrast, the degradation of cytochrome P-450 by TCPO strikingly decreased ethoxyresorufin deethylase and to a lesser extent p-nitroanisole demethylase. A reaction mechanism is proposed for the TCPO mediated degradation of cytochrome P-450 in vitro.

A key role for Pax7 transcripts in determination of muscle and nerve cells.

Embryonic teratocarcinoma mouse cells (P19) and embryonic NIH3T3 fibroblasts were induced chemically to differentiate along neurogenic or myogenic lineages. The expression profiles of Pax7 alternate transcripts were then assessed by RNA isolation and RT-PCR. Only two transcripts, Pax7b and Pax7d, were expressed in the neurogenic lineage. By contrast, in adult skeletal muscle, four transcripts, Pax7a-d, were expressed in the myogenic lineage. Moreover, P19 cells were shown to undergo neural cell differentiation when stably transfected with a single Pax7 transcript, PAX7b, generated from human skeletal muscle. Our results suggest a key role for Pax7 transcripts in lineage determination.

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.

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.

Inhibition of rat GSH S-transferases by ethylene dibromide.

Incubation of ethylene dibromide (EDB) (37 mM) with a mixture of rat hepatic cytosol GSH S-transferases at 25 degrees resulted in diminished activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB). The loss of both activities followed pseudo first order kinetics with a rate constant of 0.13 +/- 0.03 min-1. The concentration of EDB required for half maximal loss of enzymic activity towards CDNB was 3.2 mM. Removal of EDB from the enzyme by lyophilization or gel filtration did not result in the return of activity towards CDNB. GSH partially prevented the loss of activity, but could not reverse the loss. EDB decreased the activity of forms A(YbYb) and C(YbYb) of rat liver GSH S-transferases but not of forms AA(YcYc), B(YaYc) or B(YaYa). It is concluded that EDB inhibits forms A and C of the GSH S-transferases via a mechanism not involving suicide inhibition.

Variegate porphyria in South Africa, 1688-1996--new developments in an old disease.

Variegate porphyria, an autosomal dominant inherited trait resulting in decreased activity of protoporphyrinogen oxidase, the penultimate haem biosynthetic enzyme, is characterised clinically by photosensitive skin disease and a propensity to acute neurovisceral crises. The disease has an exceptionally high frequency in South Africa, owing to a founder effect. The specific mutation in the protoporphyrinogen oxidase gene sequence which represents this founder gene has been identified. Genetic diagnosis is therefore now possible in families in whom the gene defect is known. However, the exact nature and degree of activity of the porphyria can only be determined by detailed quantitative biochemical analysis of excreted porphyrins. The relative contributions of the acute attack and the skin disease to the total disease burden of patients with variegate porphyria is not static, and in South Africa there have been significant changes over the past 25 years, with fewer patients presenting with acute attacks, leaving a greater proportion to present with skin disease or to remain asymptomatic with the diagnosis being made in the laboratory. The most common precipitating cause of the acute attack of VP is administration of porphyrinogenic drugs. Specific suppression of haem synthesis with intravenous haem arginate is the most useful treatment of a moderate or severe acute attack. Although cutaneous lesions are limited to the sun-exposed areas, management of the skin disease of VP remains inadequate.