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Biomedical subjects

D M Hunt

Publications and source records attributed to D M Hunt.

At least 73 records · Page 4Linked to original sources
PubMed1998

Molecular evolution of trichromacy in primates.

Although trichromacy in Old and New World primates is based on three visual pigments with spectral peaks in the violet (SW, shortwave), green (MW, middlewave) and yellow-green (LW, longwave) regions of the spectrum, the underlying genetic mechanisms differ. The SW pigment is encoded in both cases by an autosomal gene and, in Old World primates, the MW and LW pigments by separate genes on the X chromosome. In contrast, there is a single polymorphic X-linked gene in most New World primates with three alleles coding for spectrally distinct pigments. The one reported exception to this rule is the New World howler monkey that follows the Old World system of separate LW and MW genes. A comparison of gene sequences in these different genetic systems indicates that the duplication that gave rise to the separate MW and LW genes of Old World primates is more ancient than that in the howler monkey. In addition, the amino acid sequences of the two howler monkey pigments show similarities to the pigments encoded by the polymorphic gene of other New World primates. It would appear therefore that the howler monkey gene duplication arose after the split between New and Old World primates and was generated by an unequal crossover that placed two different forms of the New World polymorphic gene on to a single chromosome. In contrast, the lack of identity at variable sites within the New and Old World systems argues for the origin of the separate genes in Old World primates by the duplication of a single form of the gene followed by divergence to give spectrally distinct LW and MW pigments. In contrast, the similarity in amino acid variation across the tri-allelic system of New World primates indicates that this polymorphism had a single origin in New World primates. A striking feature of all these pigments is the use of a common set of substitutions at three amino acid sites to achieve the spectral shift from MW at around 530 nm to LW at around 560 nm. The separate origin of the trichromacy in New and Old World primates would indicate that the selection of these three sites is the result of convergent evolution, perhaps as a consequence of visual adaptation in both cases to foraging for yellow and orange fruits against a green foliage.

Alouatta↗
PubMed1998

High frequency of persistent hyperplastic primary vitreous and cataracts in p53-deficient mice.

In order to investigate whether the p53 gene product plays a role in normal eye development, age matched p53-deficient mice and wild-type controls were sacrificed from day 2 to day 21 after birth. Eyes were paraffin-embedded and sectioned. Serial sections were taken at the level of the tunica vasculosa lentis and the hyaloid artery. The terminal dUTP nick-end labelling technique (TUNEL) was used to detect the number of cells displaying DNA fragmentation within these structures. Eyes were also prepared for scanning electron microscopy and resin embedded for semi-thin sections. Adult wild-type mice and p53-deficient mice were examined ophthalmoscopically in vivo. Ophthalmoscopical examination of mice completely deficient in p53 revealed them to be normal except for the persistence of the hyaloid vasculature, a structure that normally regresses during eye development. In adult animals there was also a high frequency of cataracts. Using morphological assessment and TUNEL we could show that in normal mice, regression of the primary vitreous, which includes the hyaloid artery, the vasa hyaloidea propria as well as the tunica vasculosa lentis, occurs via apoptotic cell death within 5 - 6 weeks after birth. The number of TUNEL-positive cells within these structures was significantly reduced in the p53-deficient mice in which parts of the hyaloid vasculature persisted and developed into a fibro-vascular retrolental plaque analogous to persistent hyperplastic primary vitreous (PHPV) described in humans. As in humans, PHPV in mice resulted in the development of cataracts. We have identified a role for p53-dependent apoptosis in the regression of the hyaloid vasculature and tunica vasculosa lentis. Our results provide further evidence for the importance of p53 in normal development and provide the first detailed evidence of its role in postnatal development in remodelling the developing eye.

Age Factors↗
PubMed1998

Immune responses limit adenovirally mediated gene expression in the adult mouse eye.

In order to investigate the immunological consequences of gene transfer to the eye using viral vectors, adenovirus carrying a lacZ reporter gene (AV.LacZ) was injected either subretinally, subconjunctivally or into the anterior chamber of three groups of adult mice: immunocompetent or transiently immunosuppressed BALB/c mice and congenic immunodeficient nude mice. Adenovirally mediated lacZ expression persisted for approximately 3 weeks following injection of the vector into the anterior chamber, retina or extra ocular tissues of the conjuctiva of BALB/c mice. It appears that T cell-mediated immune responses limit the duration of AV-mediated ocular gene expression in adult mice since lacZ gene expression was detected for at least 15 weeks in T cell-deficient BALB/c nude mice, although the level of transgene expression decreased with time. Since intra-ocular AV-mediated gene expression was not significantly longer than extra-ocular expression, it appears that the eye is not normally immune-privileged with respect to viral vectors. Inflammatory cells were detected in the vitreous after anterior chamber injection and in the retina after subretinal injection of adenovirus. The presence of both CD4+ and CD8+ T cells was established by immunophenotyping. Reinjection of BALB/c mice resulted in rapid decline in reporter gene expression, but successful readministration was possible in the case of immunodeficient nude mice. However, after transient depletion of T cells, achieved by intraperitoneal injection of both CD8- and CD4-specific antibodies, the duration of expression in BALB/c mice was longer in the eye (at least 12 weeks, again with decrease in level over time), than in extra-ocular tissues (8 weeks) provided the animal was not reinjected with virus raising the possibility of partial ocular immune-privilege after transient immunosuppression.

Adenoviridae↗
PubMed1998

Co-injection of adenovirus expressing CTLA4-Ig prolongs adenovirally mediated lacZ reporter gene expression in the mouse retina.

There is growing interest in gene delivery to the eye in order to develop gene therapy for the many ocular disorders which may be amenable to this approach. To date, recombinant adenoviruses (AV) have been the main vector used for gene delivery to anterior and posterior segments in animal models. As with delivery to other organs, immune responses to vector and transgene limit the duration of expression in the eye. Using an E1-deleted adenoviral vector carrying a lacZ reporter gene, we have previously demonstrated that a T cell-mediated immune response reduces the level of intra-ocular transgene expression over time and limits it to around 3 weeks in mice. This report describes a strategy for prolonging gene expression by blocking the B7-CD28 interactions between antigen presenting cells (APC) and T cells in order to prevent the costimulatory signals required for T cell survival and proliferation. This was achieved by the co-injection of AV encoding a secreted immunomodulatory molecule (CTLA4-Ig) which consists of the extra-cellular domain of mouse CTLA4 fused to the Fc region of human IgG. Subretinal co-injection of AV encoding beta galactosidase with AV encoding CTLA4-Ig results in prolonged expression in retinal cells compared with subretinal injection of only adenovirus encoding beta galactosidase.

Abatacept↗
PubMed1998

Absence of p53 delays apoptotic photoreceptor cell death in the rds mouse.

PURPOSE: This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. METHODS: A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. RESULTS: The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. CONCLUSIONS: We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.

Animals↗
PubMed1998

Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy.

The dominant cone-rod dystrophy gene CORD6 has previously been mapped to within an 8 cM interval on chromosome 17p12-p13. The retinal-specific guanylate cyclase gene (RETGC-1), which maps to within this genetic interval and previously was implicated in Leber's congenital amaurosis, was screened for mutations within this family and in a panel of small families and individuals with various cone and cone- rod dystrophy phenotypes. A missense mutation (E837D) was identified in affected members of the CORD6 family, as well as a second missense mutation (R838C) in three other families with dominant cone-rod dystrophy. RETGC-1 is only the fourth gene to be implicated in cone-rod dystrophy and this is the first report of dominant mutations in this gene.

Adolescent↗
PubMed1998

Phenotype of a British North Carolina macular dystrophy family linked to chromosome 6q.

AIMS: To document the phenotype of an autosomal dominant macular dystrophy diagnosed as having North Carolina macular dystrophy (NCMD) in this British family, and to verify that the disease locus corresponds with that of MCDR1 on chromosome 6q. METHODS: 37 family members were examined and the phenotype characterised. DNA samples from the affected members, 19 unaffected and five spouses, were used to perform linkage analysis with six microsatellite marker loci situated within the MCDR1 region of chromosome 6q. RESULTS: Every affected family member had lesions characteristic of NCMD, which developed early in life and usually remain stable thereafter. Although fundus changes are evident in the periphery, all tests revealed that functional loss is restricted to the macula. Some patients with large macular lesions had good visual acuity with fixation at the edge of the lesion at 5 degrees eccentricity. Significant linkage to the MCDR1 locus on chromosome 6q was obtained with three marker loci, with a maximum lod score of 5.9 (q = 0.00) obtained with D6S249. CONCLUSION: This family has the typical phenotype NCMD, and the causative gene was linked to the disease locus (MCDR1) on chromosome 6q. Early onset and localisation of the disease to the central macula allow specialisation of eccentric retina in some eyes with resultant good visual acuity.

Adolescent↗
PubMed1998

Assessment of the interphotoreceptor matrix proteoglycan-1 (IMPG1) gene localised to 6q13-q15 in autosomal dominant Stargardt-like disease (ADSTGD), progressive bifocal chorioretinal atrophy (PBCRA), and North Carolina macular dystrophy (MCDR1).

We have recently characterised the genomic organisation of a novel interphotoreceptor matrix proteoglycan, IMPG1, and have mapped the gene locus to chromosome 6q13-q15 by fluorescence in situ hybridisation. As the interphotoreceptor matrix (IPM) is thought to play a critical role in retinal adhesion and the maintenance of photoreceptor cells, it is conceivable that a defect in one of the IPM components may cause degenerative lesions in retinal structures and thus may be associated with human retinopathies. By genetic linkage analysis, several retinal dystrophies including one form of autosomal dominant Stargardt-like macular dystrophy (STGD3), progressive bifocal chorioretinal atrophy (PBCRA), and North Carolina macular dystrophy (MCDR1) have previously been localised to a region on proximal 6q that overlaps the IMPG1 locus. We have therefore assessed the entire coding region of IMPG1 by exon amplification and subsequent single stranded conformational analysis in patients from 6q linked multigeneration families diagnosed with PBCRA and MCDR1, as well as a single patient from an autosomal dominant STGD pedigree unlinked to either of the two known STGD2 and STGD3 loci on chromosomes 13q and 6q, respectively. No disease associated mutations were identified. In addition, using an intragenic polymorphism, IMPG1 was excluded by genetic recombination from both the PBCRA and the MCDR1 loci. However, as the autosomal dominant Stargardt-like macular dystrophies are genetically heterogeneous, other forms of this disorder, in particular STGD3 previously linked to 6q, may be caused by mutations in IMPG1.

Adult↗
PubMed1998

Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator.

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been associated with a number of male reproductive problems, including testis abnormalities and a reduction in germ cell quality and number. To establish at least one site of functional CFTR expression in the testis, we subjected cultured Sertoli cells to analysis of message, protein, and channel activity for CFTR. With reverse transcription-polymerase chain reaction, we obtained evidence for the presence of CFTR RNA when CFTR primers were used with RNA from cultured Sertoli cells. Western analysis performed with both anti-R and anti-C domain CFTR antibodies revealed immunoreactive material in extracts from primary Sertoli cell cultures that seemed consistent with CFTR previously identified in other cells and tissues. This led us to perform more detailed studies using the whole cell arrangement of the patch-clamp technique. Application of the membrane-soluble cAMP analog, 8-chlorophenyl-thio-cAMP, resulted in the activation of a Cl- current that displayed a permeability sequence of Br- > I- > or = Cl- and was blocked by diphenylamine-2-carboxylate and glibenclamide. In addition, a 13-pS conductance Cl- channel was measured in excised membrane patches exposed to the catalytic subunit of protein kinase A. When taken together, our findings of evidence of CFTR message, immunoreactive material that appeared consistent with CFTR, and Cl- channels with properties similar to those reported for CFTR provide strong evidence that Sertoli cells express a functional CFTR-like protein. The presence of CFTR in these cells may be needed to maintain the specific nutritional and fluid balance in the seminiferous tubule that is vital for normal spermatogenesis.

Animals↗
PubMed1998

The rhodopsin gene of the cuttlefish Sepia officinalis: sequence and spectral tuning.

The cephalopod molluscs are a group of invertebrates that occupy a wide range of oceanic photic environments. They are an ideal group of animals, therefore, in which to study the evolution of rhodopsin. The cDNA sequence of the rhodopsin gene of the cuttlefish Sepia officinalis (L.) (Sub-class Coleoidea, Order Sepiida) is presented, together with an analysis of the structure of the gene. A proline-rich C terminus is present; this structure is characteristic of cephalopod rhodopsins. In common with all invertebrate opsins studied so far, the equivalent site to the counterion in vertebrate opsins is occupied by an aromatic amino acid. An intron is present that splits codon 107, in contrast to the intronless rhodopsin gene in two species of myopsid squid. A spectral tuning model involving substitutions at only three amino acid sites is proposed for the spectral shifts between the rhodopsins of Sepia officinalis, three species of squid and Paroctopus defleini.

Amino Acid Sequence↗
PubMed1998

Vitreous treatment of retinal pigment epithelial cells results in decreased expression of FGF-2.

PURPOSE: Changes in gene expression were investigated after treatment of cultured human retinal pigment epithelial (RPE) cells with vitreous. This may have implications for proliferative diseases such as proliferative vitreoretinopathy. METHODS: Cells were cultured in the presence or absence of human vitreous, and gene expression was examined using the differential display polymerase chain reaction technique. Differentially expressed RNAs were cloned, screened for differential expression, and sequenced. The expression of one of these RNAs (that for fibroblast growth factor [FGF]-2/basic FGF) was examined by in situ hybridization and ribonuclease protection assays. The level of FGF-2 protein was examined by immunoblot analysis. The effects of adding FGF-2 to cells cultured in the presence of vitreous were examined. RESULTS: Treatment of low passage human RPE cells with 25% vitreous resulted in the epithelial-to-fibroblast-like morphologic changes reported by others and in the decreased expression of FGF-2 mRNA and FGF-2 protein. Addition of FGF-2 to cultures at the same time as addition of vitreous prevented some of the effects of vitreous on these cells. CONCLUSIONS: Vitreous treatment of RPE cells in culture results in decreased expression of FGF-2 mRNA and protein. Because supplementation of FGF-2 prevents some of the vitreous-mediated effects, this may indicate that modulation of FGF-2 levels by the vitreous may play an important role in the phenotypic changes seen in RPE cells exposed to vitreous.

Cells, Cultured↗
PubMed1997

The rod and green cone opsins of two avian species, the budgerigar, Melopsittacus undulatus, and the mallard duck, Anas platyrhynchus.

The genes for the rod and rod-like green cone opsins in two avian species, the budgerigar, Melopsittacus undulatus, and the mallard duck, Anas platyrhynchus, are identified on the basis of amino acid identity with the equivalent chicken sequences and their placement into a single phylogenetic clade with the rod and rod-like green cone opsin genes from other vertebrate species. Since the two bird species studied are taxonomically quite distinct, this would indicate that this rod-like green cone opsin gene, although absent in mammals, is common in the Aves. The two avian pigments differ consistently at site 122, consistent with the reported role of this site in determining the rate of metarhodopsin II formation and decay in rod and cone pigments. Candidate sites are identified to compensate for the known spectral effects of substitution at this site.

Amino Acid Sequence↗
PubMed1997

The number of triplet repeats in five brain-expressed loci with CAG repeats is not associated with schizophrenia.

We have previously shown that large expansions of CAG (CTG) triplets are associated both with schizophrenia itself and with an early age-at-onset of the disease. However, the repeat expansion detection (RED) method used did not provide a chromosomal location for the expanded region(s) (Morris et al., 1995). In a further study of our schizophrenic and control patients, we have now examined the length of the repeated sequence in five loci that are expressed in brain and are known to contain CAG repeat regions (Li et al., 1993). No enlarged repeat regions were identified; it is unlikely therefore that expansions at any of these five loci can account for expansions of up to 136 triplets identified by the RED method.

Age of Onset↗
PubMed1997

Mechanisms of wavelength tuning in the rod opsins of deep-sea fishes.

The main object of this study was to investigate the molecular basis for changes in the spectral sensitivity of the visual pigments of deep-sea fishes. The four teleost species studied, Hoplostethus mediterraneus, Cataetyx laticeps, Gonostoma elongatum and Histiobranchus bathybius, are phylogenetically distant from each other and live at depths ranging from 500 to almost 5000 m. A single fragment of the intronless rod opsin gene was PCR-amplified from each fish and sequenced. The wavelength of peak sensitivity for the rod visual pigments of the four deep-sea species varies from 483 nm in H. mediterraneus and G. elongatum to 468 nm in C. laticeps. Six amino acids at sites on the inner face of the chromophore-binding pocket formed by the seven transmembrane a-helices are identified as candidates for spectral tuning. Substitutions at these sites involve either a change of charge, or a gain or loss of a hydroxyl group. Two of these, at positions 83 and 292, are consistently substituted in the visual pigments of all four species and are likely to be responsible for the shortwave sensitivity of the pigments. Shifts to wavelengths shorter than 480 nm may involve substitution at one or more of the remaining four sites. None of the modifications found in the derived sequences of these opsins suggest functional adaptations, such as increased content of hydroxyl-bearing or proline residues, to resist denaturation by the elevated hydrostatic pressures of the deep sea. Phylogenetic evidence for the duplication of the rod opsin gene in the Anguilliform lineage is presented.

Adaptation, Physiological↗