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Biomedical subjects

D C Shields

Publications and source records attributed to D C Shields.

At least 55 records · Page 3Linked to original sources
PubMed1998

Upregulation of calpain activity and expression in experimental allergic encephalomyelitis: a putative role for calpain in demyelination.

The degradation of myelin proteins has been implicated in destabilization of the myelin sheath in autoimmune demyelinating diseases such as multiple sclerosis (MS). In order to investigate the role of calcium-activated neutral proteinase (calpain), which degrades myelin proteins, the activity and expression (translational and transcriptional) of this enzyme were examined in spinal cords of Lewis rats with experimental allergic encephalomyelitis (EAE), an animal model of MS. In addition to calpain, the translational expression of calpastatin (endogenous inhibitor of calpain) and extent of neurofilament (NFP) and myelin protein degradation were evaluated via Western blotting in controls and rats with EAE. The transcriptional expression of millicalpain, microcalpain, and calpastatin as examined by RT-PCR was not significantly increased in EAE. However, calpain translational expression was increased by 206. 5% while the levels of 68 kDa NFP and myelin-associated glycoprotein were decreased by 42.9 and 39.7%, respectively, in animals with EAE compared to controls. Calpastatin isoforms (180, 110, 80, and 68 kDa) were significantly increased in EAE as well. The findings of increased activity and translational expression of calpain in EAE suggest a major role for this enzyme in myelinolysis associated with autoimmune demyelinating diseases.

Animals↗
PubMed1998

Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis.

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Animals↗
PubMed1998

Increased calpain expression in experimental demyelinating optic neuritis: an immunocytochemical study.

Since calcium activated neutral proteinase (calpain) is present in the central nervous system (CNS) and degrades myelin proteins, this endopeptidase has been suggested to play a role in myelin destruction in demyelinating diseases such as multiple sclerosis (MS). In the present study, calpain immunocytochemical expression was examined in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS and optic neuritis. To identify cells expressing calpain, we labeled rat optic nerve sections for calpain with a polyclonal myelin calpain antibody and with monoclonal antibodies for glial (GFAP, OX42) and inflammatory (CD2, ED2, ED1, IFN-gamma) cell-specific markers. The results showed increased calpain expression in microglia (OX42) and infiltrating macrophages (ED1,2) in EAE compared to normal controls. Astrocytes constitutively expressed calpain in controls and acute EAE. Reactive astrocytes in EAE located in or near inflammatory foci, exhibited markedly increased calpain expression. Most T cells in acute EAE showed low level calpain expression while activated IFN-gamma-producing lymphocytes in inflammatory foci exhibited elevated levels of calpain expression. Thus, our results demonstrate increased calpain expression (at transcriptional and/or translational levels) in a rat model of optic neuritis. A role for calpain in myelin destruction during optic neuritis may be relevant to the pathogenesis of this disorder.

Animals↗
PubMed1998

Putative role of calpain in the pathophysiology of experimental optic neuritis.

Since myelin proteins are degraded in autoimmune demyelinating diseases such as optic neuritis, proteinases are believed to participate in myelinolysis. Calpain (calcium activated neutral proteinase) degrades myelin proteins at physiological pH and is found in glial and inflammatory cells involved in demyelination. To examine the putative role of calpain in myelinolysis, the activity and expression (translational and transcriptional) of this enzyme and endogenous inhibitor, calpastatin were examined in optic nerves of Lewis rats with experimental allergic encephalomyelitis (EAE), an animal model of optic neuritis. Calpain activity was examined via Western blotting by measuring the extent of myelin protein degradation and calpain-specific fodrin proteolysis in optic nerves from controls versus rats with experimental optic neuritis. RT-PCR studies demonstrated no significant change in millicalpain, microcalpain, or calpastatin expression at the mRNA level in optic nerves from animals with experimental optic neuritis compared to controls. However, myelin associated glycoprotein (MAG) levels were decreased by 25.5% while calpain translational expression and calpain-autolyzed fodrin levels were increased by 72.1% and 462.8% respectively, in experimental optic neuritis compared to controls. Translational expression of calpastatin isoforms (80, 68 and 55 KD) was not significantly different in rats with experimental optic neuritis compared to controls. Thus, increased activity and translational expression of calpain in experimental optic neuritis suggests this proteinase may participate in the degradation of myelin and cytoskeletal proteins in demyelinating diseases such as optic neuritis.

Animals↗
PubMed1998

Preliminary evidence of an association between bipolar disorder in females and the catechol-O-methyltransferase gene.

Catechol-O-methyltransferase (COMT) catalyses the methylation, and hence the inactivation, of catecholamines including the neurotransmitters dopamine and noradrenaline. There is evidence implicating COMT as a candidate gene for a number of neuropsychiatric conditions including bipolar disorder. A long recognized population variation in COMT activity exists and it has recently been established that variation in enzyme activity results from a polymorphic genetic variation within the COMT gene which can be readily assayed as a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A collection of 60 Irish bipolar I probands have been genotyped together with their parents. Tests comparing transmitted and non-transmitted alleles provide no evidence that the polymorphism contributes to a susceptibility to bipolar disorder within the sample as a whole. However, amongst female bipolar I probands (n = 30) there was a tendency for the low-activity allele of COMT to be preferentially transmitted. Furthermore, a re-examination of an Irish case-control sample resulted in a similar observation amongst female bipolar I sufferers and pooling the data sets strengthened the findings.

Alleles↗
PubMed1997

Lack of evidence for a major locus for bipolar disorder in the pericentromeric region of chromosome 18 in Irish pedigrees.

Seven families, multiply affected by bipolar mood disorder, have been collected from the Irish population and have been genotyped with microsatellite markers from the pericentromeric region of chromosome 18, a region that has been implicated as a site for a susceptibility gene for this relative common psychiatric disorder. The families significantly excluded linkage of bipolar disorder to this region under various models. Although the data provided no evidence of linkage heterogeneity among families, the number of families investigated may be too small to exclude completely the possibility of linkage in a small number of families.

Adult↗
PubMed1997

Molecular evidence for an ancient duplication of the entire yeast genome.

Gene duplication is an important source of evolutionary novelty. Most duplications are of just a single gene, but Ohno proposed that whole-genome duplication (polyploidy) is an important evolutionary mechanism. Many duplicate genes have been found in Saccharomyces cerevisiae, and these often seem to be phenotypically redundant. Here we show that the arrangement of duplicated genes in the S. cerevisiae genome is consistent with Ohno's hypothesis. We propose a model in which this species is a degenerate tetraploid resulting from a whole-genome duplication that occurred after the divergence of Saccharomyces from Kluyveromyces. Only a small fraction of the genes were subsequently retained in duplicate (most were deleted), and gene order was rearranged by many reciprocal translocations between chromosomes. Protein pairs derived from this duplication event make up 13% of all yeast proteins, and include pairs of transcription factors, protein kinases, myosins, cyclins and pheromones. Tetraploidy may have facilitated the evolution of anaerobic fermentation in Saccharomyces.

Chromosomes, Fungal↗
PubMed1997

Acute phase proteins in salmonids: evolutionary analyses and acute phase response.

Inflammation induces dramatic changes in the biosynthetic profile of the liver, leading to increased serum concentrations of positive acute phase (AP) proteins and decreased concentrations of negative AP proteins. Serum amyloid A (SAA) and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are major AP proteins: their serum levels can rise by 1000-fold, indicating that they play a critical role in defense and/or the restoration of homeostasis. We have cloned SAA and a SAP-like pentraxin from salmonid fish species. The salmonid SAA shares approximately 70% amino acid identity with mammalian AP SAA. When salmonids are challenged with an AP stimulus, i.e., Aeromonas salmonicida, SAA responds dramatically as a major AP reactant. The salmonid pentraxin shows approximately 40% amino acid identity to both mammalian SAP and CRP. Evolutionary analysis suggests the presence of only a single such protein in teleosts and lower animal species. Surprisingly, the salmonid pentraxin behaves as a negative AP reactant, reminiscent of the SAP-like Syrian hamster female protein, in that hepatic mRNA concentrations decline to 50% of prestimulus levels. This study reinforces the hypothesis that SAA induction is an essential and universal feature of the vertebrate AP response and that it represents part of an ancient host defense system. Conversely, the species-dependent heterogeneity of pentraxin expression during the vertebrate AP response supports the possibility that its most important ancestral (and perhaps present) function is not related to its AP behavior.

Acute-Phase Proteins↗
PubMed1997

Calcium-mediated neurofilament protein degradation in rat optic nerve in vitro: activity and autolysis of calpain proenzyme.

In this study, we examined calcium-mediated degradation of a neurofilament protein (NFP), and autolytic activation of calpain in Lewis rat optic nerve in vitro. After incubation with calcium, homogenized optic nerve samples were analysed by SDS-PAGE in association with ECL immunoblot techniques. 68 kD NFP, calpain, and calpastatin antibodies were used for identification of the respective proteins. The extent of calcium-mediated 68 kD NFP degradation compared to EGTA controls, served to quantify calpain activity, while the extent of calpain autolysis measured the activation of the enzyme. A progressive loss of 68 kD NFP was observed at 15 min (42.1%), 1 hr (52.7%) and 6 hr (73.4%) incubation periods compared to EGTA controls. The immunoreactive calpain bands showed progressive autolysis after 15 min (26.6%), 1 hr (31.4%) and 6 hr (43.4%) incubations. We also found degradation of low molecular weight isoforms of calpastatin (43 kD and 27 kD) in the presence of calcium compared to controls. These results indicate that calpain is present in optic nerve in its inactive form but when calcium is added, it undergoes autolysis and becomes active. Thus, active calpain is capable of degrading endogenous substrates (e.g. cytoskeletal and myelin proteins) and may promote the degeneration of optic nerve in optic neuritis.

Animals↗
PubMed1997

Accelerated evolution of sites undergoing mRNA editing in plant mitochondria and chloroplasts.

The selective constraints influencing mRNA editing in plant organelles are largely unknown. To investigate these, we compared patterns of editing between monocot and dicot mitochondrial mRNA. On average, 24% of sites that are edited form C to U in one species have been substituted during evolution by a genomic T in the other: this is four times the rate of evolution seen at nonedited synonymously variable C residues. A similar, but weaker trend (not statistically significant) is seen at sites edited in chloroplast mRNA. The elevated substitution rate does not appear to be a consequence of a higher mutability of the trinucleotide motif (T-C-purine) associated with editing. nor to be a result of reverse transcription from mature mRNA. Selection to replace the genomic C with a T may account for the accelerated evolution, either due to elimination of inefficient transcripts and protein products or as a consequence of the prior loss of components outside the edit site which are necessary for editing; the latter hypothesis is supported by the frequent loss of editing without genomic mutation at third codon positions. Whatever the cause, the rapid rate of evolution indicates that editing confers little selective advantage at most sites.

Base Sequence↗
PubMed1996

Mapping genes within a YAC by computer-assisted interpretation of partial restriction digestions.

Partial restriction digestion is used to map restriction sites and the location of genes within yeast artificial chromosomes (YACs). Locus-specific probes are hybridised to the partially digested YAC DNA and the fragments to which they hybridise are compared with the pattern of partial digestion products that include each map region. A least squares criterion is presented which allows for error in fragment length determination. This rapidly defines the most likely location of a marker within the restriction map and permits the combination of results from digestions with different restriction enzymes. Approximate confidence intervals may be assigned to gene locations, and tests of goodness-of-fit of the data may be performed. Since the number of erroneously matched fragments increases in proportion to the square of the number of sites, denser maps are not necessarily more informative. Simulations indicate that the optimal number of internal restriction sites given typical experimental error (1% of YAC length) is about five sites; the associated broad support interval (on average one third of YAC length) may be reduced by combining results from different enzyme digestions. Application of a computer implementation of this model to experimental data showed that the model fitted well, and estimates of location were found to be consistent with other evidence.

Animals↗
PubMed1996

Homocysteine and risk of premature coronary heart disease. Evidence for a common gene mutation.

BACKGROUND: Plasma homocysteine levels are modulated by nutritional and genetic factors, among which is the enzyme methylenetetrahydrofolate reductase (MTHFR). A common defective (thermolabile) variant of this enzyme is causally associated with elevated plasma homocysteine, itself an independent risk factor for coronary heart disease. METHODS AND RESULTS: To examine the hypothesis that the allele (T) that codes for the thermolabile defect increases the risk of coronary heart disease, we studied 111 patients with clinical and objective investigational evidence of coronary heart disease and 105 control subjects. The frequencies of the thermolabile defect (T) in patients and control subjects were measured, and the prevalence of elevated plasma total homocysteine according to genotype was assessed. The frequency of the defective allele was higher in patients than in control subjects with an OR of 1.6 (95% CI, 1.1 to 2.4; P = .02). The OR in the coronary heart disease group for the homozygous TT genotype was 2.9 (95% CI, 1.2 to 7.2; P = .02); 17% of patients and 7% of control subjects had the TT genotype. Plasma total homocysteine levels were significantly associated with disease status, a relationship that matched the strength of the association between disease and homozygous inheritance of the defective enzyme. CONCLUSIONS: Homozygotes for the defective allele (T) are at increased risk of premature coronary heart disease. MTHFR, which modulates basal plasma homocysteine concentration, is folate dependent, and dietary supplementation or fortification with folic acid may reduce plasma homocysteine levels and consequent coronary risk in a significant proportion of the general population.

Alanine↗
PubMed1996

Evolution of hemopoietic ligands and their receptors. Influence of positive selection on correlated replacements throughout ligand and receptor proteins.

The rates of amino acid replacement in cytokines and their receptors are high and vary considerably. To determine whether this reflects the action of positive selection, rates of nonsynonymous DNA substitution were examined and found to exceed the synonymous substitution rate in certain exons of rodent IL-3, granulocyte-macrophage stimulating factor, and IL-4. To determine the extent to which positive selection could account for correlations between the amino acid replacement rates of hemopoietins and their receptors, rates were examined in various domains: the correlation with ligand rate was not confined to the ligand-binding domain of the receptor, but extended into the transmembrane and cytoplasmic domains and even to leader peptide domains of both ligand and receptor. As the majority of these replacements are unlikely to be strongly advantageous, different levels of both positive and purifying selection contribute to the extensive variation in hemopoietin/receptor evolutionary rates. Changes in a few residues critical for ligand-receptor interaction may be followed by changes of lesser selective importance in both molecules: replacements of growth hormone residues that form hydrogen or salt bridges with the receptor occur in lineages in which there are many concurrent replacements. A ligand/receptor rate correlation is not found between the seven-transmembrane receptors and their ligands, whose mature forms are often short and completely conserved. This study predicts that a minority of concurrent evolutionary changes in hemopoietins and their receptors reflect directly compensatory changes.

Amino Acids↗
PubMed1996

Wallaby serum amyloid A protein: cDNA cloning, sequence and evolutionary analysis.

A serum amyloid A (SAA) clone was isolated from a Tammar wallaby cDNA library, the most distantly related mammalian species for which an SAA has been described to date. The clone predicts a premolecule of 127 amino acids with good homology to other mammalian SAAs, and consists of an 18 residue leader peptide and a mature protein of 109 amino acids. Evolutionary analysis at both the protein and nucleotide level indicate that the wallaby SAA clone clusters with the acute phase SAAs. However, as the SAA superfamily has undergone concerted evolution it is not possible to determine at this point which acute phase SAA it is most like. The grouping of wallaby SAA inside the acute phase SAA cluster demonstrates that at least some of the duplication events giving rise to multiple acute phase genes occurred prior to the divergence of the eutherian and metatherian mammals.

Amino Acid Sequence↗
PubMed1996

Analysis of the thyrotropin receptor as a candidate gene in familial Graves' disease.

Familial clustering of Graves' disease indicates a genetic etiology. Searches for genetic factors additional to the known human leukocyte antigen (HLA) association have implicated the gene for the TSH receptor (TSHR). We analyzed the linkage and association among three recently described microsatellite markers within the TSHR introns in Graves' disease in large multiply affected Welsh and English families (223 members, 44 affected individuals). Linkage analysis under a dominant model strongly rejected the hypothesis that TSHR is linked to Graves' disease in these families (lod score = -4.53). More detailed analyses also failed to provide evidence for linkage; these included combined segregation and linkage analysis, correction for HLA-DR3 status, allowance for the levels of thyroid autoantibodies in unaffected pedigree members, consideration of a recessive model for the disease, and linkage disequilibrium between disease and marker alleles. We also considered the possibility of a genetic heterogeneity of Graves' disease and thus analyzed separately the different families with a similar result. Although these results cannot eliminate a minor role of the TSHR gene locus in the genetics of Graves' disease, they argue against it being a major genetic determinant in this pathology.

Adolescent↗
PubMed1995

The evolution of haematopoietic cytokine/receptor complexes.

The evolutionary expansion of the haematopoietic cytokines and their receptors is characterized by the duplication of both cytokines and receptors. A systematic analysis of primary sequence homology indicates that receptors for gp130-associated cytokines group into signal transducing and non-signal transducing receptors. This observation is consistent with the evolution of the interleukins 6, 11 and 12, granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M, and the ciliary neurotrophic factor complexes from a common ancestral complex which included a homodimer of gp130-like signalling receptors and an interleukin 6 receptor-like non-signalling receptor. Alterations in the components of the complex are proposed to have arisen by receptor duplication and divergence to allow signal transduction via a LIF receptor/gp130 heterodimer, and loss of the non-signalling receptor component in the G-CSF and the LIF lineage. The short-chain haematopoietins and their receptors do not group clearly, although interleukins 4 and 13 grouped together, as did 2 and 10. Internal duplication of the ligand-binding domain appears to have occurred independently in three separate lineages. These observations have implications for the classification of cytokines and receptors, and for the modelling by homology of their structures and interactions.

Amino Acid Sequence↗