Visual system evolution and the nature of the ancestral snake.

The dominant hypothesis for the evolutionary origin of snakes from 'lizards' (non-snake squamates) is that stem snakes acquired many snake features while passing through a profound burrowing (fossorial) phase. To investigate this, we examined the visual pigments and their encoding opsin genes in a range of squamate reptiles, focusing on fossorial lizards and snakes. We sequenced opsin transcripts isolated from retinal cDNA and used microspectrophotometry to measure directly the spectral absorbance of the photoreceptor visual pigments in a subset of samples. In snakes, but not lizards, dedicated fossoriality (as in Scolecophidia and the alethinophidian Anilius scytale) corresponds with loss of all visual opsins other than RH1 (λmax 490-497 nm); all other snakes (including less dedicated burrowers) also have functional sws1 and lws opsin genes. In contrast, the retinas of all lizards sampled, even highly fossorial amphisbaenians with reduced eyes, express functional lws, sws1, sws2 and rh1 genes, and most also express rh2 (i.e. they express all five of the visual opsin genes present in the ancestral vertebrate). Our evidence of visual pigment complements suggests that the visual system of stem snakes was partly reduced, with two (RH2 and SWS2) of the ancestral vertebrate visual pigments being eliminated, but that this did not extend to the extreme additional loss of SWS1 and LWS that subsequently occurred (probably independently) in highly fossorial extant scolecophidians and A. scytale. We therefore consider it unlikely that the ancestral snake was as fossorial as extant scolecophidians, whether or not the latter are para- or monophyletic.

Genetic linkage of cone-rod retinal dystrophy to chromosome 19q and evidence for segregation distortion.

Inherited retinal dystrophies are the most common cause of childhood blindness in the developed world. Cone-rod retinal dystrophies are severe examples of this group of disorders. Analysis of a large cone-rod dystrophy pedigree suggested that inheritance within the family was influenced by meiotic drive (p = 0.008), a rare segregation distortion in human genetics. Two-point linkage analysis showed significant linkage with three markers mapping to chromosome 19q. Multipoint analysis gave a maximum lod score of 10.08 (theta = 0.05) distal to D19S47. Cone-rod dystrophy is therefore assigned to 19q13.1-q13.2 and a new candidate locus for other retinal dystrophies is identified.

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Identification and characterization of visual pigments in caecilians (Amphibia: Gymnophiona), an order of limbless vertebrates with rudimentary eyes.

In comparison with the other amphibian orders, the Anura (frogs) and Urodela (salamanders), knowledge of the visual system of the snake-like Gymnophiona (caecilians) is relatively sparse. Most caecilians are fossorial with, as far as is known any surface activity occurring mainly at night. They have relatively small, poorly developed eyes and might be expected to possess detectable changes in the spectral sensitivity of their visual pigments. Microspectrophotometry was used to determine the spectral sensitivities of the photoreceptors in three species of caecilian, Rhinatrema bivittatum, Geotrypetes seraphini and Typhlonectes natans. Only rod opsin visual pigment, which may be associated with scotopic (dim light) vision when accompanied by other 'rod-specific' components of the phototransduction cascade, was found to be present. Opsin sequences were obtained from the eyes of two species of caecilian, Ichthyophis cf. kohtaoensis and T. natans. These rod opsins were regenerated in vitro with 11-cis retinal to give pigments with spectral sensitivity peaks close to 500 nm. No evidence for cone photoreception, associated with diurnal and colour vision, was detected using molecular and physiological methods. Additionally, visual pigments are short-wavelength shifted in terms of the maximum absorption of light when compared with other amphibian lineages.

The chemistry of John Dalton's color blindness.

John Dalton described his own color blindness in 1794. In common with his brother, he confused scarlet with green and pink with blue. Dalton supposed that his vitreous humor was tinted blue, selectively absorbing longer wavelengths. He instructed that his eyes should be examined after his death, but the examination revealed that the humors were perfectly clear. In experiments presented here, DNA extracted from his preserved eye tissue showed that Dalton was a deuteranope, lacking the middlewave photopigment of the retina. This diagnosis is shown to be compatible with the historical record of his phenotype, although it contradicts Thomas Young's belief that Dalton was a protanope.

Adaptive evolution of color vision genes in higher primates.

The intron 4 sequences of the three polymorphic alleles at the X-linked color photo-pigment locus in the squirrel monkey and the marmoset reveal that the alleles in each species are exceptionally divergent. The data further suggest either that each triallelic system has arisen independently in these two New World monkey lineages, or that in each species at least seven deletions and insertions (14 in the two species) in intron 4 have been transferred and homogenized among the alleles by gene conversion or recombination. In either case, the alleles in each species apparently have persisted more than 5 million years and probably have been maintained by overdominant selection.

A novel connexin50 mutation associated with congenital nuclear pulverulent cataracts.

PURPOSE: To screen for mutations of connexin50 (Cx50)/GJA8 in a panel of patients with inherited cataract and to determine the cellular and functional consequences of the identified mutation. METHODS: All patients in the study underwent a full clinical examination and leucocyte DNA was extracted from venous blood. The GJA8 gene was sequenced directly. Connexin function and cellular trafficking were examined by expression in Xenopus oocytes and HeLa cells. RESULTS: Screening of the GJA8 gene identified a 139 G to A transition that resulted in the replacement of aspartic acid by asparagine (D47N) in the coding region of Cx50. This change co-segregated with cataract among affected members of a family with autosomal dominant nuclear pulverulent cataracts. While pairs of Xenopus oocytes injected with wild type Cx50 RNA formed functional gap junction channels, pairs of oocytes injected with Cx50D47N showed no detectable intercellular conductance. Co-expression of Cx50D47N did not inhibit gap junctional conductance of wild type Cx50. In transiently transfected HeLa cells, wild type Cx50 localised to appositional membranes and within the perinuclear region, but Cx50D47N showed no immunostaining at appositional membranes with immunoreactivity confined to the cytoplasm. Incubation of HeLa cells transfected with Cx50D47N at 27 degrees C resulted in formation of gap junctional plaques. CONCLUSIONS: The pulverulent cataracts present in members of this family are associated with a novel GJA8 mutation, Cx50D47N, that acts as a loss-of-function mutation. The consequent decrease in lens intercellular communication and changes associated with intracellular retention of the mutant connexin may contribute to cataract formation.

A novel GJA8 mutation is associated with autosomal dominant lamellar pulverulent cataract: further evidence for gap junction dysfunction in human cataract.

PURPOSE: To identify the gene responsible for autosomal dominant lamellar pulverulent cataract in a four-generation British family and characterise the functional and cellular consequences of the mutation. METHODS: Linkage analysis was used to identify the disease locus. The GJA8 gene was sequenced directly. Functional behaviour and cellular trafficking of connexins were examined by expression in Xenopus oocytes and HeLa cells. RESULTS: A 262C>A transition that resulted in the replacement of proline by glutamine (P88Q) in the coding region of connexin50 (Cx50) was identified. hCx50P88Q did not induce intercellular conductance and significantly inhibited gap junctional activity of co-expressed wild type hCx50 RNA in paired Xenopus oocytes. In transfected cells, immunoreactive hCx50P88Q was confined to the cytoplasm but showed a temperature sensitive localisation at gap junctional plaques. CONCLUSIONS: The pulverulent cataract described in this family is associated with a novel GJA8 mutation and has a different clinical phenotype from previously described GJA8 mutants. The cataract likely results from lack of gap junction function. The lack of function was associated with improper targeting to the plasma membrane, most probably due to protein misfolding.

Panton-Valentine leukocidin-secreting Staphylococcus aureus causing severe musculoskeletal sepsis in children. A new threat.

Panton-Valentine leukocidin secreted by Staphylococcus aureus is known to cause severe skin, soft tissue and lung infections. However, until recently it has not been described as causing life-threatening musculoskeletal infection. We present four patients suffering from osteomyelitis, septic arthritis, widespread intravascular thrombosis and overwhelming sepsis from proven Panton-Valentine leukocidin-secreting Staphylococcus aureus. Aggressive, early and repeated surgical intervention is required in the treatment of these patients. The Panton-Valentine leukocidin toxin not only destroys host neutrophils, immunocompromising the patient, but also increases the risk of intravascular coagulopathy. This combination leads to widespread involvement of bone with glutinous pus which is difficult to drain, and makes the delivery of antibiotics and eradication of infection very difficult without surgical intervention.

Update on paediatric ACL injuries.

In the last two decades there has been an increase in the incidence of anterior cruciate ligament (ACL) injuries in children. This may be due to increased awareness, more participation in high demand contact and non-contact sports at an earlier age and better diagnosis as a result of better imaging. A review of the literature suggests that the long-term results of non-operative treatment are poor. While the short to medium-term results of ACL reconstruction in children are encouraging, the long-term results are unknown. In this review, the current trends in the management of paediatric ACL injuries are discussed with particular emphasis on the natural history, surgical techniques, the effect of surgery on the growth plate and complications.

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.

Chromosomal localization, genomic organization and evolution of the genes encoding human phosphatidylinositol transfer protein membrane-associated (PITPNM) 1, 2 and 3.

Eukaryotic proteins containing a phosphatidylinositol transfer (PITP) domain can be divided into two groups, one consisting of small soluble 35-kDa proteins and the other those that are membrane-associated and show sequence similarities to the Drosophila retinal degeneration B (rdgB) protein. The rdgB protein consists of four domains, an amino terminal PITP domain, a Ca2+-binding domain, a transmembrane domain and a carboxyl terminal domain that interacts with the protein tyrosine kinase PYK2. Three mammalian phosphatidylinositol transfer protein membrane-associated genes (PITPNM1, 2 and 3) with homology to Drosophila rdgB have previously been described and shown to be expressed in the mammalian retina. These findings and the demonstration that the rdgB gene plays a critical role in the invertebrate phototransduction pathway have led to the mammalian genes being considered as candidate genes for human eye diseases. In order to facilitate the analysis of these genes we have used radiation hybrid mapping and fluorescence in situ hybridization to localize the PITPNM2 and 3 genes to human chromosomes 12p24 and 17p13 respectively and hybrid mapping to confirm the localization of PITPNM1 to chromosome 11q13. We have also determined the genomic organization of both the soluble and membrane-associated Drosophila and human PITP domain-containing genes. Phylogenetic analysis indicates that the two groups arose by gene duplication that occurred very early in animal evolution.

Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an aromatic polyketide synthase.

BACKGROUND: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-beta-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain 'Streptomyces maritimus' deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides. RESULTS: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from 'S. maritimus' has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis. CONCLUSIONS: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.

A detailed study of the phenotype of an autosomal dominant cone-rod dystrophy (CORD7) associated with mutation in the gene for RIM1.

AIM: To characterise the phenotype of an autosomal dominant cone-rod dystrophy (CORD7) associated with the Arg844His mutation in RIM1. METHODS: Eight members of a four generation, non-consanguineous British family were examined clinically and underwent electrophysiological testing, automated dark adapted perimetry, dark adaptometry, colour vision assessment, colour fundus photography, fundus fluorescein angiography (FFA), and fundus autofluorescence (AF) imaging. RESULTS: The majority of affected individuals described a progressive deterioration of central vision, night vision, and peripheral visual field usually between the third and fourth decades. The visual acuity ranged from 6/6 to 3/60. Colour vision testing showed mild to moderate dyschromatopsia in the majority of individuals. Fundus changes comprised a range of macular appearances varying from mild retinal pigment epithelial (RPE) disturbance to extensive atrophy and pigmentation. In some individuals retinal vessels were attenuated and in two subjects peripheral areas of retinal atrophy were present. An absent or severely reduced PERG was detected in all subjects, indicative of marked macular dysfunction. Full field ERG showed abnormal rod and cone responses. AF imaging revealed decreased macular AF centrally surrounded by a ring of increased AF in the majority of individuals. "Bull's eye" lesions were present in two individuals, comprising of a ring of decreased perifoveal AF bordered peripherally and centrally by increased AF. Photopic sensitivity testing demonstrated elevated central visual field thresholds with additional superior greater than inferior peripheral field loss. There were rod and cone sensitivity reductions in the central and peripheral visual fields, with the inferior retina being more affected than the superior. CONCLUSIONS: The detailed phenotype is described of the autosomal dominant cone-rod dystrophy, CORD7, which is associated with a point mutation in RIM1, a gene encoding a photoreceptor synaptic protein. The pattern of disease progression and long term visual outcome facilitates improved genetic counselling and advice on prognosis. Such phenotypic data will be invaluable in the event of future therapy.

A detailed phenotypic study of "cone dystrophy with supernormal rod ERG".

AIMS: To characterise the detailed phenotype of "cone dystrophy with supernormal rod ERG" in a case series of 10 patients. METHODS: 10 affected patients were examined clinically and underwent colour fundus photography, with nine undergoing detailed electrophysiological testing. Five patients were assessed further with fundus autofluorescence (AF) imaging, automated photopic and dark adapted perimetry, and dark adaptometry. Detailed colour vision assessment was performed in six subjects. Blood samples were taken from four patients for DNA extraction and mutation screening of NR2E3 was undertaken. RESULTS: The onset of symptoms was in the first and second decades of life. Subjects presented with reduced central vision and marked photophobia. All individuals were myopic and colour vision testing revealed severely reduced colour discrimination predominantly along the red-green axes; tritan colour vision was relatively well preserved. Nyctalopia is a later feature of the disorder. Funduscopy and AF imaging revealed a range of macular appearances. There was electrophysiological evidence of marked macular dysfunction, reduced and delayed cone responses, and supernormal and delayed rod responses. Photopic and dark adapted perimetry revealed central scotomata with widespread peripheral sensitivity loss. No disease causing sequence variants in NR2E3 were identified. CONCLUSIONS: The largest case series to date has been described of the clinical, psychophysical and electrophysiological characteristics of this unusual cone dystrophy with supernormal rod responses. Electrophysiological data were consistent with a post-phototransduction, but pre-inner nuclear layer, site of dysfunction. While the definitive diagnosis can only be made with electrophysiological testing, several characteristics that may increase suspicion of this diagnosis are presented.

The genetics of inherited macular dystrophies.

The inherited macular dystrophies comprise a heterogeneous group of disorders characterised by central visual loss and atrophy of the macula and underlying retinal pigment epithelium (RPE). The different forms of macular degeneration encompass a wide range of clinical, psychophysical and histological findings. The complexity of the molecular basis of monogenic macular disease is now beginning to be elucidated with the identification of many of the disease-causing genes. Age related macular degeneration (ARMD), the leading cause of blind registration in the developed world, may also have a significant genetic component to its aetiology. Genes implicated in monogenic macular dystrophies are good candidate susceptibility genes for ARMD, although to date, with the possible exception of ABCA4, none of these genes have been shown to confer increased risk of ARMD. The aim of this paper is to review current knowledge relating to the monogenic macular dystrophies, with discussion of currently mapped genes, chromosomal loci and genotype-phenotype relationships. Inherited systemic disorders with a macular dystrophy component will not be discussed.

Mapping of a novel locus for achromatopsia (ACHM4) to 1p and identification of a germline mutation in the alpha subunit of cone transducin (GNAT2).

OBJECTIVE: To determine the molecular basis for achromatopsia using autozygosity mapping and positional candidate gene analysis. DESIGN AND METHODS: A large consanguineous Pakistani family containing six subjects with autosomal recessive complete achromatopsia was ascertained. After excluding linkage to the two known achromatopsia genes (CNGA3 and CNGB3), a genome wide linkage screen was undertaken. RESULTS: Significant linkage was detected to a 12 cM autozygous segment between markers D1S485 and D1S2881 on chromosome 1p13. Direct sequence analysis of the candidate gene GNAT2 located within this interval identified a frameshift mutation in exon 7 (c842_843insTCAG; M280fsX291) that segregated with the disease. CONCLUSIONS: The GNAT2 gene codes for cone alpha-transducin, the G protein that couples the cone pigments to cGMP-phosphodiesterase in phototransduction. Although cone alpha-transducin has a fundamental role in cone phototransduction, mutations in GNAT2 have not been described previously. Since mutations in the CNGA3 gene may cause a variety of retinal dystrophies (complete and incomplete achromatopsia and progressive cone dystrophy), GNAT2 mutations may also prove to be implicated in other forms of retinal dystrophy with cone dysfunction.

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.

Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.

The adhesive proteins of the desmosome type of cell junction consist of two types of cadherin found exclusively in that structure, the desmogleins and desmocollins, coded by two closely linked loci on human chromosome 18q12.1. Recently we have identified a mutation in the DSG1 gene coding for desmoglein 1 as the cause of the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK) in which affected individuals have marked hyperkeratotic bands on the palms and soles. In the present study we present the complete exon-intron structure of the DSG1 gene, which occupies approximately 43 kb, and intron primers sufficient to amplify all the exons. Using these we have analysed the mutational changes in this gene in five further cases of SPPK. All were heterozygotic mutations in the extracellular domain leading to a truncated protein, due either to an addition or deletion of a single base, or a base change resulting in a stop codon. Three mutations were in exon 9 and one in exon 11, both of which code for part of the third and fourth extracellular domains, and one was in exon 2 coding for part of the prosequence of this processed protein. This latter mutation thus results in the mutant allele synthesising only 25 amino acid residues of the prosequence of the protein so that this is effectively a null mutation implying that dominance in the case of this mutation was caused by haploinsufficiency. The most severe consequences of SPPK mutations are in regions of the body where pressure and abrasion are greatest and where desmosome function is most necessary. SPPK therefore provides a very sensitive measure of desmosomal function.

Cloning vectors for the production of proteins in Dictyostelium discoideum.

We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high-copy-number plasmid Ddp2, expression cassettes consisting of the strong, constitutive actin (act15) or the inducible discoidin (disI gamma) promoters, a translational start codon upstream from a multiple cloning site and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) His residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High synthesis levels were obtained for each vector; up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products.