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D M Hunt

Publications and source records attributed to D M Hunt.

At least 19 recordsLinked to original sources
PubMed1999

Meniscal movement. An in-vivo study using dynamic MRI.

We present the first study in vivo of meniscal movement in normal knees under load. Using an open MR scanner, allowing imaging in physiological positions in near to real-time, 16 young footballers were scanned moving from full extension to 90 degrees flexion in the sagittal and coronal planes. Excursion of the meniscal horns, radial displacement and meniscal height were measured. On weight-bearing, the anterior horn of the medial meniscus moves through a mean of 7.1 mm and the posterior horn through 3.9 mm, with 3.6 mm of mediolateral radial displacement. The height of the anterior horn increases by 2.6 mm and that of the posterior horn by 2.0 mm. The anterior horn of the lateral meniscus moves 9.5 mm and the posterior horn 5.6 mm, with 3.7 mm of radial displacement. The height of the anterior horn increases by 4.0 mm, and that of the posterior horn by 2.4 mm. In non-weight-bearing, the anterior horn of the medial meniscus moves 5.4 mm and the posterior horn 3.8 mm, with 3.3 mm of radial displacement. The anterior horn of the lateral meniscus moves 6.3 mm, and the posterior horn 4.0 mm, with 3.4 mm of radial displacement. The most significant differences between weight-bearing and non-weight-bearing were the movement and vertical height of the anterior horn of the lateral meniscus.

Adolescent↗
PubMed1999

Expression of antioxidant protective proteins in the rat retina during prenatal and postnatal development.

PURPOSE: In retinopathy of prematurity, capillary growth in the retina is attenuated. Subsequent cyclic elevation of oxygen levels leads to renewed capillary growth that may eventually result in retinal detachment. It is hypothesized that the sensitivity of the premature retina to oxidative shock results from the absence of antioxidant protective proteins. METHODS: The expression of heme oxygenase-1, metallothionein, superoxide dismutase, and catalase mRNAs was measured in retinas of rats from 6 days before birth to 4 days after birth using in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction with Southern blot analysis. RESULTS: Superoxide dismutase mRNA was expressed to a similar extent at all time points. Metallothionein mRNA expression, which was high at embryonic days (E) 16 and 18, decreased to low levels by the time of birth and remained low at least until 4 days after birth. Catalase mRNA expression was low until birth and increased until at least postnatal day 4. Heme oxygenase-1 mRNA showed low expression at E16 and E18, increased before birth, and then diminished. CONCLUSIONS: Four antioxidant protein mRNAs showed very different patterns of expression in the rat retina. Two of these proteins, heme oxygenase-1 and catalase, were expressed at relatively low levels until approximately the time of birth. The former is important in protection against heme-mediated generation of reactive oxygen species, whereas the latter protects against hydrogen peroxide-generated damage. As a result of the low expression of these mRNAs, and presumably the proteins encoded by them, the premature rat (and probably the premature human) is likely to be born without a full complement of antioxidant defenses.

Animals↗
PubMed1998

Isolation and chromosomal localization of two human CDP-diacylglycerol synthase (CDS) genes.

Phototransduction in Drosophila is a phosphoinositide-mediated signaling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signaling. cds mutants develop light-induced retinal degeneration. As part of a search for novel genes that may be involved in eye disease in human, using Drosophila phototransduction genes as a model system, two human CDP-diacylglycerol synthase genes (CDS1 and CDS2) were cloned and sequenced. Radiation hybrid panel mapping and fluorescence in situ hybridization were used to localize the genes to chromosomes 4q21 and 20p13. As yet, no known retinal diseases map to either of these regions.

Amino Acid Sequence↗
PubMed1998

The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus).

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

Amino Acid Sequence↗
PubMed1998

Adeno-associated virus gene transfer to mouse retina.

Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.

Animals↗
PubMed1998

Expression of the protective proteins hemopexin and haptoglobin by cells of the neural retina.

The blood-retinal barrier, consisting of retinal pigment epithelial cells and retinal endothelial cells, prevents hemopexin and haptoglobin, anti-oxidant protective plasma proteins normally synthesized by the liver, from entering the neural retina. If present, these proteins must, therefore, be made locally. The cell types within the retina in which hemopexin and haptoglobin mRNAs are made have been investigated. RNA was extracted from both the neural retina and pigment epithelium obtained by dissection of human donor eyes as well as from cultured pigment epithelial and photoreceptor cells. The mRNAs for both haptoglobin and hemopexin were detected, using reverse-transcriptase polymerase chain reaction, in the neural retina and cultured photoreceptors but not in pigment epithelial cells. The cellular location of these mRNAs was determined using in situ hybridization of sections of human retina which revealed that haptoglobin mRNA was located principally in the photoreceptor cells, cells of the inner nuclear layer and some cells of the ganglion cell layer. Hemopexin mRNA, previously shown to be made in the human neural retina (Hunt et al., 1996. Journal of Cellular Physiology 168: 71-80), is expressed by most of the cells of neural retina including the photoreceptors and, notably, the ganglion cells.

Antioxidants↗
PubMed1998

Molecular genetics of spectral tuning in New World monkey color vision.

Although most New World monkeys have only one X-linked photopigment locus, many species have three polymorphic alleles at the locus. The three alleles in the squirrel monkey and capuchin have spectral peaks near 562, 550, and 535 nm, respectively, and the three alleles in the marmoset and tamarin have spectral peaks near 562, 556, and 543 nm, respectively. To determine the amino acids responsible for the spectral sensitivity differences among these pigment variants, we sequenced all exons of the three alleles in each of these four species. From the deduced amino acid sequences and the spectral peak information and from previous studies of the spectral tuning of X-linked pigments in humans and New World monkeys, we estimated that the Ala --> Ser, Ile --> Phe, Gly --> Ser, Phe --> Tyr, and Ala --> Tyr substitutions at residue positions 180, 229, 233, 277, and 285, respectively, cause spectral shifts of about 5, -2, -1, 8, and 15 nm. On the other hand, the substitutions His --> Tyr, Met --> Val or Leu, and Ala --> Tyr at positions 116, 275, and 276, respectively, have no discernible spectral tuning effect, though residues 275 and 276 are inside the transmembrane domains. Many substitutions between Val and Ile or between Val and Ala have occurred in the transmembrane domains among the New World monkey pigment variants but apparently have no effect on spectral tuning. Our study suggests that, in addition to amino acid changes involving a hydroxyl group, large changes in residue size can also cause a spectral shift in a visual pigment.

Amino Acid Substitution↗
PubMed1998

Post-receptoral mechanisms of colour vision in New World primates.

Diurnal platyrrhines, both di- and trichromats, have magnocellular (M-) and parvocellular (P-) retinal ganglion cells which are morphologically very similar to those found in catarrhines. Catarrhine central P ganglion cells contact single midget bipolar cells, which contact single cones. Physiological recordings of retinal ganglion cells of dichromatic Cebus monkeys showed very similar cell properties to the catarrhine macaque, except that P ganglion cells lacked colour-opponency. We describe the presence of single-headed midget bipolar cells in the Cebus retina. These midget bipolar cells have axon terminal sizes in the same range as the dendritic tree sizes of P ganglion cells as far as 2 mm of retinal eccentricity. This result supports the view that, as in catarrhines, central P ganglion cells of platyrrhines receive input from single midget bipolar cells which in turn, receive input from single cones. This finding is consistent with the idea that a P pathway with one-to-one connectivity was present in the anthropoid ancestor before the divergence between catarrhines and platyrrhines.

Action Potentials↗
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

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↗