Assignment of claudin-1 (CLDN1) to human chromosome 3q28-->q29 with somatic cell hybrids.
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Chromosome Mapping↗
Biomedical subjects
D M Hunt
Publications and source records attributed to D M Hunt.
The orthopaedic management of peripheral ischaemia in meningococcal septicaemia in children.
Between March 1993 and February 1999, 14 children aged from eight months to 14.75 years were admitted to the paediatric intensive-care unit with meningococcal septicaemia in association with severe peripheral ischaemia. Of these, 13 were operated upon, eight of whom had early fasciotomies. Five children died. Of the nine survivors, one had no amputations while in the other eight 14 limb segments were amputated. We review the case histories and propose a protocol for the early management of these children.
Adolescent↗
Clinical outcome of congenital talipes equinovarus diagnosed antenatally by ultrasound.
Congenital talipes equinovarus is a common anomaly which can now be diagnosed prenatally on a routine ultrasound scan at 20 weeks of gestation. Prenatal counselling is increasingly offered to parents with affected fetuses, but it is difficult to counsel parents if there is a chance that the fetus may not have talipes. Our study correlates the prenatal ultrasound findings of 14 infants diagnosed as having unilateral or bilateral talipes during their routine 20-week ultrasound scan with their clinical findings at birth and the treatment received. No feet diagnosed as talipes on the ultrasound scan were completely normal at birth and therefore there were no true false-positive results. One foot graded as normal at 20 weeks was found to have a mild grade-1 talipes at birth, but did not require treatment other than simple stretches. A total of 32% of feet required no treatment and so could be considered functional false-positive results on the scan. Serial casting was required by 13% of feet and surgical treatment by 55%. The severity of the talipes is difficult to establish before birth. A number of patients are likely to need surgical treatment, but a proportion will have talipes so mildly that no treatment will be required. In counselling parents at 20 weeks, orthopaedic surgeons need to know whether or not there is a small chance that the ultrasound diagnosis could be wrong and also that the talipes may be so mild that the foot will not require treatment.
Casts, Surgical↗
Clustered cadherin genes: a sequence-ready contig for the desmosomal cadherin locus on human chromosome 18.
We describe the assembly of a cosmid and PAC contig of approximately 700 kb on human chromosome 18q12 spanning the DSC and DSG genes coding for the desmocollins and desmogleins. These are members of the cadherin superfamily of calcium-dependent cell adhesion proteins present in the desmosome type of cell junction found especially in epithelial cells. They provide the strong cell-cell adhesion generated by this type of cell junction for which expression of both a desmocollin and a desmoglein is required. In the autoimmune skin diseases pemphigus foliaceous and pemphigus vulgaris (PV), where the autoantigens are, respectively, encoded by the DSG1 and DSG3 genes, severe areas of acantholysis (cell separation), potentially life-threatening in the case of PV, are evident. Dominant mutations in the DSG1 gene causing striate palmoplantar keratoderma result in hyperkeratosis of the skin on the parts of the body where pressure and abrasion are greatest, viz., on the palms and soles. These genes are also candidate tumor suppressor genes in squamous cell carcinomas and other epithelial cancers. We have screened two chromosome 18-specific cosmid libraries by hybridization with previously isolated YAC clones and DSC and DSG cDNAs, and a whole genome PAC library, both by hybridization with the YACs and by screening by PCR using cDNA sequences and YAC end sequence. The contigs were extended by further PCR screens using STSs generated by vectorette walking from the ends of the cosmids and PACs, together with sequence from PAC ends. Despite screening of two libraries, the cosmid contig still had four gaps. The PAC contig filled these gaps and in fact covered the whole locus. The positions of 45 STSs covering the whole of this region are presented. The desmocollin and desmoglein genes, which are about 30-35 kb in size, are quite well separated at approximately 20-30 kb apart and are arranged in two clusters, one DSC cluster and one DSG cluster, which are transcribed outward from the interlocus region. The order of the genes is correlated with the spatial order of gene expression in the developing mouse embryo, and this, and previous transgenic experiments, suggests that long-range genetic elements that coordinate expression of these genes may be present. The complete bacterial clone contig described in this paper is thus a resource not only for future sequencing but also for investigations into the control of expression of these clustered genes.
Bacteriophage P1↗
Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.
Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.
Amino Acid Sequence↗
Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in Malacosteus niger, a deep-sea dragon fish with far red bioluminescence.
Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin pigment pair with lambda max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments (lambda max > 550 nm) observed in two other red light producing stomiids. The presence of only a single opsin gene in the M. niger genome was confirmed by molecular and cladistic analysis. To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M. niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation.
Animals↗
Visual pigments and oil droplets in the retina of a passerine bird, the canary Serinus canaria: microspectrophotometry and opsin sequences.
The visual receptors of the passeriform bird Serinus canaria, the canary, have been examined microspectrophotometrically and the sequences of the opsins determined. Rods have a maximum absorbance (lambda max) at 506 nm. Four spectral classes of single cone are present: long-wave-sensitive (LWS) containing a photopigment with lambda max at 569 nm, middle-wave-sensitive (MWS) with lambda max at 505 nm, short-wave-sensitive (SWS) with lambda max at 442 nm, and ultraviolet-sensitive (UVS) with lambda max at about 366 nm. Double cones possess the 569-nm pigment in both members. Typical combinations of photopigment and oil droplet occur in most cone classes. An ambiguity exists in the oil droplet of the single LWS cones. In some birds, LWS cones are paired with an R-type droplet, whereas in the majority of canaries the LWS pigment is paired with a droplet similar to the P-type of double cones. Mechanisms of spectral tuning within each opsin class are discussed.
Animals↗
N-terminal deletion in a desmosomal cadherin causes the autosomal dominant skin disease striate palmoplantar keratoderma.
The N-terminal extracellular domain of the cadherins, calcium-dependent cell adhesion molecules, has been shown by X-ray crystallography to be involved in two types of interaction: lateral strand dimers and adhesive dimers. Here we describe the first human mutation in a cadherin present in desmosome cell junctions that removes a portion of this highly conserved first extracellular domain. The mutation, in the DSG1 gene coding for a desmoglein (Dsg1), results in the deletion of the first and much of the second beta-strand of the first cadherin repeat and part of the first Ca2+-binding site, and would be expected to compromise strand dimer formation. It causes a dominantly inherited skin disease, striate palmoplantar keratoderma (SPPK), mapping to chromosome 18q12.1, in which affected individuals have marked hyperkeratotic bands on the palms and soles. In a three generation Dutch family with SPPK, we have found a G-->A transition in the 3" splice acceptor site of intron 2 of the DSG1 gene which segregated with the disease phenotype. This causes aberrant splicing of exon 2 to exon 4, which are in-frame, with the consequent removal of exon 3 encoding part of the prosequence, the mature protein cleavage site and part of the first extracellular domain. This mutation emphasizes the importance of this part of the molecule for cadherin function, and of the Dsg1 protein and hence desmosomes in epidermal function.
Amino Acid Sequence↗
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↗
The use of dynamic interventional MRI in developmental dysplasia of the hip.
Conventional methods of imaging in the investigation of developmental dysplasia of the hip all have disadvantages, either in definition or in exposure to radiation. We describe a new open-configuration MR scanner which is unique in that it allows anaesthesia and access to the patient within the imaging volume for surgical procedures and application of casts. We performed 13 scans in eight anaesthetised infants. Dynamic imaging revealed two dislocated hips which were then visualised during reduction. Hip spicas were applied without removing the patient from the scanner. In one hip, an adductor tenotomy was carried out. In all patients, stressing the hips during dynamic imaging allowed an assessment of stability. This was particularly useful in two hips in which an analysis of stability in different positions facilitated the planning of femoral osteotomies. This method of imaging provides new and important information. It has great potential in the investigation of developmental dysplasia of the hip and, with ultrasound, may allow management without the need for radiography.
Anesthesia, General↗
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↗
The evolution of trichromatic color vision by opsin gene duplication in New World and Old World primates.
Trichromacy in all Old World primates is dependent on separate X-linked MW and LW opsin genes that are organized into a head-to-tail tandem array flanked on the upstream side by a locus control region (LCR). The 5' regions of these two genes show homology for only the first 236 bp, although within this region, the differences are conserved in humans, chimpanzees, and two species of cercopithecoid monkeys. In contrast, most New World primates have only a single polymorphic X-linked opsin gene; all males are dichromats and trichromacy is achieved only in those females that possess a different form of this gene on each X chromosome. By sequencing the upstream region of this gene in a New World monkey, the marmoset, we have been able to demonstrate the presence of an LCR in an equivalent position to that in Old World primates. Moreover, the marmoset sequence shows extensive homology from the coding region to the LCR with the upstream sequence of the human LW gene, a distance of >3 kb, whereas homology with the human MW gene is again limited to the first 236 bp, indicating that the divergent MW sequence identifies the site of insertion of the duplicated gene. This is further supported by the presence of an incomplete Alu element on the upstream side of this insertion point in the MW gene of both humans and a cercopithecoid monkey, with additional Alu elements present further upstream. Therefore, these Alu elements may have been involved in the initial gene duplication and may also be responsible for the high frequency of gene loss and gene duplication within the opsin gene array. Full trichromacy is present in one species of New World monkey, the howler monkey, in which separate MW and LW genes are again present. In contrast to the separate genes in humans, however, the upstream sequences of the two howler genes show homology with the marmoset for at least 600 bp, which is well beyond the point of divergence of the human MW and LW genes, and each sequence is associated with a different LCR, indicating that the duplication in the howler monkey involved the entire upstream region. [The sequence data described in this paper have been submitted to GenBank under accession nos. AF155218, AF156715, and AF156716.]
Animals↗
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↗
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↗
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↗
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↗
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↗
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↗