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Biomedical subjects

B McInnes

Publications and source records attributed to B McInnes.

At least 19 recordsLinked to original sources
PubMed2000

Effectiveness of a bed positioning program for treating older adults with knee contractures who are institutionalized.

BACKGROUND AND PURPOSE: Although contractures in patients in long-term care institutions are an important issue, there have been only a few studies that have evaluated interventions for contractures. The purpose of this study was to determine the effectiveness of a bed positioning program (BPP) for the treatment of patients with knee flexion contractures. SUBJECTS: Sixteen patients with a high level of cognitive and functional impairment (mean age=82 years, SD=6.48, range=71-93) in a chronic care hospital participated in the study. METHODS: The BPP consisted of stretching a patient's knee into extension and then securing and maintaining the position for a period of 40 minutes, 4 times per week. Participants were randomly assigned to 2 groups (n=8 in each group). One group received a BPP for 8 weeks, followed by 8 weeks of no intervention. The other group received the intervention in the reverse order. Once a week, participants were assessed for range of knee extension, knee pain, and skin integrity. RESULTS: Twelve participants completed the study. There was no improvement in participants' range of knee extension during the intervention period. Overall, there was no difference in mean range of knee extension between the intervention period and the no-intervention period. CONCLUSION AND DISCUSSION: The results of this study do not support the use of a BPP for treating patients with knee flexion contractures.

Aged↗
PubMed1999

Analysis of aneuploidy for chromosomes 13, 21, X and Y by multicolour fluorescence in situ hybridisation (FISH) in a 47,XYY male.

The frequency of aneuploid sperm was assessed by fluorescence in situ hybridisation (FISH) in a 47,XYY male previously studied by sperm karyotyping. A total of 20,021 sperm were studied: 10,017 by two-colour FISH for chromosomes 13 and 21 and 10,002 by three-colour FISH for the sex chromosomes using chromosome 1 as an autosomal control for diploidy and lack of hybridisation. Results were compared with more than 500,000 sperm from 18 normal men. The frequencies of X-bearing (49.4%) and Y-bearing sperm (49.8%) were not significantly different from 50% as shown in our sperm karyotyping study. There was no significant increase in the frequency of diploid sperm compared with control donors. There was a significant increase in the frequency of disomy for chromosome 13 (p < 0.0001) and XY disomy (p = 0.0008) compared with control donors. However, since the frequency of disomy was 0.40% for chromosome 13 and 0.55% for XY disomy, it is not surprising that these increases were not discovered previously in our analysis of 75 sperm karyotypes. Our results suggest that the extra Y chromosome is eliminated during spermatogenesis in the majority of cells but that there may be a small but significant increase in the frequency of aneuploid sperm in these men.

Adult↗
PubMed1998

A Pro504 --> Ser substitution in the beta-subunit of beta-hexosaminidase A inhibits alpha-subunit hydrolysis of GM2 ganglioside, resulting in chronic Sandhoff disease.

The GM2 gangliosidoses are caused by mutations in the genes encoding the alpha- (Tay-Sachs) or beta- (Sandhoff) subunits of heterodimeric beta-hexosaminidase A (Hex A), or the GM2 activator protein (AB variant), a substrate-specific co-factor for Hex A. Although the active site associated with the hydrolysis of GM2 ganglioside, as well as part of the binding site for the ganglioside-activator complex, is associated with the alpha-subunit, elements of the beta-subunit are also involved. Missense mutations in these genes normally result in the mutant protein being retained in the endoplasmic reticulum and degraded. The mutations associated with the B1-variant of Tay-Sachs are rare exceptions that directly affect residues in the alpha-active site. We have previously reported two sisters with chronic Sandhoff disease who were heterozygous for the common HEXB deletion allele. Cells from these patients had higher than expected levels of mature beta-protein and residual Hex A activity, approximately 20%. We now identify these patients' second mutant allele as a C1510T transition encoding a beta-Pro504 --> Ser substitution. Biochemical characterization of Hex A from both patient cells and cotransfected CHO cells demonstrated that this substitution (a) decreases the level of heterodimer transport out of the endoplasmic reticulum by approximately 45%, (b) lowers its heat stability, (c) does not affect its Km for neutral or charged artificial substrates, and (d) lowers the ratio of units of ganglioside/units of artificial substrate hydrolyzed by a factor of 3. We concluded that the beta-Pro504 --> Ser mutation directly affects the ability of Hex A to hydrolyze its natural substrate but not its artificial substrates. The effect of the mutation on ganglioside hydrolysis, combined with its effect on intracellular transport, produces chronic Sandhoff disease.

Amino Acid Substitution↗
PubMed1998

Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men.

Sperm samples from infertile men with oligozoospermia or teratozoospermia were studied by multicolour fluorescence in-situ hybridization (FISH) using DNA probes for chromosomes 13 and 21. A total of 90 809 sperm nuclei from nine infertile men and 182 799 sperm nuclei from 18 control donors were analysed. There was a highly significant increase in the frequency of spermatozoa disomic for chromosome 13 in infertile patients (0.28%) compared to control donors (0.13%) (two-tailed Z statistic P < 0.0001) and for chromosome 21 (0.48% in infertile men versus 0.37% in controls, P < 0.0001). Also there was a significantly increased frequency of diploid spermatozoa in infertile men (0.85%) compared to control donors (0.66%) (P < 0.0001). Our previous studies on these same infertile patients demonstrated increased frequencies of sperm disomy for chromosomes 1 and XY. This suggests that infertile men, who are prime candidates for intracytoplasmic sperm injection, may be at a very small increased risk of aneuploid offspring.

Adult↗
PubMed1998

Donor age and the frequency of disomy for chromosomes 1, 13, 21 and structural abnormalities in human spermatozoa using multicolour fluorescence in-situ hybridization.

The purpose of this study was to determine if a donor age effect exists for the frequency of aneuploidy and other chromosome abnormalities in human spermatozoa. Sperm samples were collected from 18 healthy men from the general population. Each individual belonged to one of six age groups (20-24, 25-29, 30-34, 35-39, 40-44, > or = 45 years) with three men in each group. Two multicolour fluorescence in-situ hybridizations were performed on spermatozoa from each donor using probes for chromosomes 13 and 21, and two chromosome 1-specific probes allowed for detection of duplications and deletions as well as disomy of chromosome 1. The abnormality frequencies and the Pearson correlation coefficients were calculated to determine if a relationship existed between donor age and the frequency of chromosome abnormalities in spermatozoa. A statistically significant association with donor age was detected for the frequency of acentric fragments of chromosome 1 (P < 0.05).

Adult↗
PubMed1997

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Acute Disease↗
PubMed1994

Genotype-phenotype pitfalls in Gaucher disease.

Gaucher disease (GD), caused by inherited deficiency of beta-glucocerebrosidase (beta-Glc, EC 3.1.2.45), is classified type I if the CNS is not involved (non-neuronopathic), type II if CNS involvement is early and rapidly progressive (acute neuronopathic), and type III if CNS involvement occurs later and is slowly progressive (subacute neuronopathic). The clinical course is not predictable by measurement of residual beta-Glc activity. Patient classification by identification of specific mutations is more promising: homozygosity for the common A5841->G (N370S) mutation invariably predicts type I; homozygosity for the T6433->C (L444P) mutation usually indicates type III (Norbottnian). Type II disease patients often carry the T6433->C allele together with a complex allele derived in part from the downstream pseudogene by crossover or gene conversion, producing a T6433->C substitution, plus 2 or 3 additional single base substitutions (fusion gene). Employing selective PCR amplification of the structural gene, we detected homozygous T6433C (L444P) point mutations in a Caucasian boy, initially classified as having GD type I, who succumbed to severe visceral GD before age 3 years. A second novel PCR procedure for discriminating between the normal gene and the fusion gene confirmed the homozygous point mutation results. Post mortem neuropathological findings showed neuronal complex lipid accumulation consistent with late-onset type III disease. Although in Norbottnian patients it is generally accepted that onset of neurological findings is delayed, patients with the L444P/L444P genotype can only be initially classified as type III with this ancestry. Other patients described sporadically elsewhere are invariably considered type I until neurological findings arise.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease.

Beta-hexosaminidase A (beta-N-acetyl-D-hexosaminidase, EC 3.2.1.5.2) is a lysosomal hydrolase composed of an alpha- and a beta-subunit. It is responsible for the degradation of GM2 ganglioside. Mutations in the HEXB gene encoded beta-subunit cause a form of GM2 gangliosidosis known as Sandhoff disease. Although this is a rare disease in the general population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16-29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5' donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compared of the intron 2 G-->A substitution and a second allele due to a 4-bp deletion in exon 7. The beta-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid screening procedure for 5' donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.

Argentina↗
PubMed1992

Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (alpha beta) and B (beta beta). The alpha subunit is encoded by the HEXA gene and the beta subunit by HEXB gene. Defects in the alpha or beta subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, AGA/GTC, to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect beta-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity.

Amino Acid Sequence↗
PubMed1992

Identification of a processed pseudogene related to the functional gene encoding the GM2 activator protein: localization of the pseudogene to human chromosome 3 and the functional gene to human chromosome 5.

The GM2 activator protein is an essential substrate cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A (EC 3.2.1.52). There have been conflicting reports as to the chromosomal localization of the gene encoding the activator. We demonstrate here that these conflicts were caused by the presence of a previously unidentified processed activator-pseudogene on chromosome 3, and we confirm a previous ELISA-based localization of the functional activator gene to chromosome 5. Our data indicate that the functional activator locus can still be considered a candidate site for defects causing some forms of spinal muscular atrophy.

Animals↗
PubMed1992

An unusual splicing mutation in the HEXB gene is associated with dramatically different phenotypes in patients from different racial backgrounds.

Sandhoff disease is caused by mutations affecting the beta subunit of lysosomal beta-hexosaminidase (EC 3.2.1.52) and displays a wide spectrum of clinical phenotypes. We report a 57-year-old patient with a very mild phenotype, although residual hexosaminidase A activity in his cultured fibroblasts was less than 3% of normal activity, a level observed in juvenile onset patients. Northern and Western blot analyses confirmed a similar low level of beta subunit-mRNA and mature beta-protein, respectively. Two mutations of the HEXB gene were identified in this patient, a partial 5' gene deletion (a null allele), and a C----T transition 8 nucleotides downstream from the intron 10/exon 11 junction affecting the splicing of the beta subunit-mRNA. In their homozygous forms, the 5' deletion has been previously shown to result in a severe infantile phenotype, and the C----T transition in a juvenile phenotype. The genotype and the low level of residual hexosaminidase A activity would be expected to produce a juvenile Sandhoff phenotype in this patient, as well as in four of his six clinically normal siblings. The biochemical basis of his mild phenotype is uncertain, but may result from genetic variations in the RNA splicing machinery.

Base Sequence↗
PubMed1991

Isolation and expression of a full-length cDNA encoding the human GM2 activator protein.

We report the construction of a cDNA clone encoding a functional GM2-activator protein. The sequence of the complete 5' end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (beta-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled GM2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.

Amino Acid Sequence↗
PubMed1991

Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells.

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.

Animals↗
PubMed1990

Structure-function studies of interferon-alpha based on random mutagenesis and expression in vitro.

An efficient procedure for random chemical mutagenesis was used to create analogs of human interferon (IFN)-alpha 4. Unique restriction enzyme sites were introduced into the human IFN-alpha 4 gene to enable cassetting of the gene for localized random mutagenesis. Single-stranded IFN-alpha 4 DNA was treated with nitrous acid, followed by second-strand synthesis using reverse transcriptase. A 72 base pair cassette spanning the coding region for amino acid residues 120 to 136 (120-136 region) was isolated and cloned into a phagemid vector adjacent to a GC-rich sequence. A DNA segment comprising the IFN-alpha 4 cassette sequence and the GC clamp was excised and electrophoresed on a denaturing gradient gel, which allowed the separation from unmutated DNA of DNA fragments with single base pair changes. DNA fragments with mobility different from that of the unmutated fragment were pooled and cloned into an expression vector. Using this procedure, mutations were found in the DNA of 48% of the clones analyzed. However, mutations at two "hot spots" accounted for 89% of these clones. Four of the IFN-alpha 4 analogs with mutations in the 120-136 region were expressed in vitro. The antiproliferative activities on human Daudi cells of most of the analogs were less than 0.2% of the activity of unmodified IFN-alpha 4, suggesting that the integrity of the carboxy terminus is important for the antiproliferative activity of human IFN-alpha 4.

Amino Acid Sequence↗
PubMed1990

Structure and distribution of an Alu-type deletion mutation in Sandhoff disease.

Sandhoff disease is a recessively inherited lysosomal storage disease resulting from a deficiency of beta-hexosaminidase activity. The enzyme occurs in two major forms, beta-hexosaminidase A, composed of an alpha- and beta-subunit and beta-hexosaminidase B, composed of two beta-subunits. Both isozyme activities are deficient in Sandhoff disease, owing to mutations of the HEXB gene encoding the common beta-subunit. We have cloned and fully characterized a deletion at the HEXB gene from fibroblasts of a patient with the infantile form of Sandhoff disease. The deletion removes approximately 16 kb of DNA including the HEXB promoter, exons 1-5 and part of intron 5. It most likely arose from recombination between two Alu sequences, with the breakpoints occurring at the midpoint between the left and right arms in each case and regenerating an intact Alu element in the deletion sequence. The deletion allele accounts for 27% of the Sandhoff mutant alleles we analyzed. Two cell lines were shown to be homozygous for the deletion and both had the infantile form of the disease. Four additional patients were compound heterozygotes with other mutations, all of whom displayed a different clinical phenotype. Finally, the mutant allele was present in different ethnic backgrounds, suggesting that it may have been subject to genetic drift.

Base Sequence↗
PubMed1989

Functional significance of amino acid residues within conserved hydrophilic regions in human interferons-alpha.

Site-directed in vitro mutagenesis was used to create analogs of human interferons (IFNs)-alpha 1 and -alpha 4. Analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate cell-free protein synthesis system. Amino acid substitutions for the highly conserved residues at positions 33, 121, 122 and 123 greatly reduced the antiviral and antiproliferative activities on human cells of IFNs-alpha 1 and -alpha 4. In general, the amino acid substitutions had much less effect on the antiviral activities on bovine, compared with human, cells. Substitutions at positions 31, 41, 42, 124, 134, 135 and 136 had little or no effect on the biological activities of the IFN analogs. The abrogation of antiviral activity resulting from amino acid substitutions for the arginine residue at position 33 suggests that this arginine residue is required for binding to the IFN-alpha receptor on the cell surface.

Amino Acid Sequence↗
PubMed1989

Structure-function studies of interferons-alpha: amino acid substitutions at the conserved residue tyrosine 123 in human interferon-alpha 1.

Analogs of human interferon-alpha 1 (IFN-alpha 1) were created in vitro by site-directed mutagenesis to investigate the structural requirements at amino acid position 123 for binding to the IFN receptor, antiviral activity, and antiproliferative activity. The tyrosine residue 123, which is conserved in all known mammalian IFNs-alpha and -beta, was replaced by each of 6 amino acids or was deleted from the protein. Several of the substitutions at position 123 partly or completely abrogated antiviral and antiproliferative activities of human IFN-alpha 1 when human or murine cells were used but not when bovine cells were used. However, with analogs in which amino acids structurally related to tyrosine, phenylalanine, or tryptophan were substituted at position 123, there was retention of antiviral and antiproliferative activities using homologous cells. Thus, although there is not an absolute requirement for tyrosine at position 123, conformational changes associated with alterations of this residue are prejudicial to the biological functions of the IFN-alpha molecule.

Amino Acids↗