Aberrant retinal tight junction and adherens junction protein expression in an animal model of autosomal dominant Retinitis pigmentosa: the Rho(-/-) mouse.

Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited diseases that are characterised by primary degeneration of rod photoreceptors and secondary degeneration of cone photoreceptors in the retina. Additional pathological changes include vascular changes and invasion of the inner retina by retinal pigment epithelial (RPE) cells. RP represents a major cause of progressive retinal disease worldwide. Using a mouse model of autosomal dominant Retinitis pigmentosa (adRP) with retinopathy induced by targeted disruption of the rhodopsin gene Rho(-/-), we have analysed the levels of expression of a range of tight and adherens junction associated proteins, in order to further elucidate the pathogenic mechanisms occurring at an early stage of this condition. Using western blot analysis and indirect immunostaining of retinal cryosections from 6-week-old mice from a C-129 background we have determined changes, if any, in the levels of expression and localisation of a series of tight and adherens junction associated proteins, including Zonula Occludens-1 (ZO-1), occludin, N-Cadherin, p120-Catenin, alpha-Catenin, gamma-Catenin, beta-Catenin, and E-Cadherin. We have found an up-regulation of the tight junction and adherens junction associated protein Zonula Occludens-1 (ZO-1) in the neural retina of 6-week-old Rho(-/-) knockout mice compared with 6-week-old Wild-Type (WT) mice. Following immunohistochemistry, however, it appears, that ZO-1, beta-Catenin and p120-Catenin expression at the Outer Limiting Membrane (OLM) of the Rho(-/-) retina is compromised, in part, compared to WT animals of the same age. We hypothesise that these retinal changes following photoreceptor cell death may contribute to the pathogenesis of adRP. Our findings of changes in the levels of expression of ZO-1 and associated adherens junction proteins beta-Catenin and p120-Catenin at the OLM in 6-week-old Rho(-/-) mice provide evidence for tight junction and adherens junction associated protein modifications in an animal model of autosomal dominant RP (adRP).

A de novo designed peptide ligase: a mechanistic investigation.

A 33-residue de novo designed peptide ligase is reported which catalyzes the template-directed condensation of suitably activated short peptides with catalytic efficiencies in excess of 10(5) ([k(cat)/K(m)]/k(uncat)). The ligase peptide, derived from natural and designed alpha-helical coiled-coil proteins, presents a surface for substrate assembly via formation of a hydrophobic core at the peptide interface. Charged residues flanking the core provide additional binding specificity through electrostatic complementarity. Addition of the template to an equimolar fragment solution results in up to 4100-fold increases in initial reaction rates. Dramatic decreases in efficiency upon mutation of charged residues or increase in ionic strength establishes the importance of electrostatic recognition to ligase efficiency. Although most of the increase in reaction efficiency is due to entropic gain from binding of substrates in close proximity, mechanistic studies with altered substrates demonstrate that the system is highly sensitive to precise ordering at the point of ligation. Taken together these results represent the first example of a peptide catalyst with designed substrate binding sites which can significantly accelerate a bimolecular reaction and support the general viability of alpha-helical protein assemblies in artificial enzyme design.

Efficient introduction of protected guanidines in boc solid phase peptide synthesis.

[reaction: see text] Reaction of primary amines with pyrazole 1 results in rapid and efficient guanidinylation, either in solution or on solid phase. The reaction affords sulfonamide-protected products required for BOC solid phase peptide synthesis (SPPS) in a single step under mild conditions. Incorporation of orthogonally protected side chain amines permits the synthesis of peptides containing arginine analogues, one of which could not be prepared by coupling of preformed amino acids.

Evaluation of human diacylglycerol kinase(iota), DGKI, a homolog of Drosophila rdgA, in inherited retinopathy mapping to 7q.

PURPOSE: To determine the genomic organization of diacylglycerol kinase(iota) and to test whether defects in this gene are present in individuals affected with autosomal dominant retinitis pigmentosa (adRP). Diacylglycerol kinase(iota) has been mapped to the RP10 locus on 7q and shows 49% sequence similarity to the Drosophila DGK2 rdgA gene. Since mutations in the DGK2 rdgA gene cause photoreceptor degeneration in Drosophila, it is possible that mutations in diacylglycerol kinase(iota) could be responsible for human retinal degeneration. METHODS: DNA sequence from genomic clones containing diacylglycerol kinase(iota) was compared with the cDNA sequence to identify intron/exon boundaries. Single-strand conformational analysis and PCR product sequencing were used to screen members of one family previously mapped to the RP10 locus and 47 small unmapped families with autosomal dominant retinitis pigmentosa. RESULTS: Diacylglycerol kinase(iota) is divided into 35 exons with the initiation codon being present in exon 2. Mutational analysis found a missense change (Lys153Phe) in three adRP families; however, it did not segregate with disease in one of the families. Silent substitutions were seen in codons 865 and 875. Intronic variation was detected in the amplifications of exons 3,5,18, 28, and 32; these do not affect splice site consensus sequences. Typing of a polymorphic variant detected in intron 31 in members of the RP10 family gave a LOD score of -4.2 at 0% recombination. CONCLUSIONS: No evidence of disease-associated mutations was found in any of the samples tested. Based on the linkage data and mutation screening, diacylglycerol kinase(iota) is excluded as a candidate for the RP10 form of adRP and cannot be a frequent cause of other forms of adRP. Mutations in diacylglycerol kinase(iota) may yet be the cause of recessive forms of retinal degeneration in humans, either in homozygous or compound heterozygous forms. The data provided here will permit testing of this hypothesis.

Retinitis pigmentosa and progressive sensorineural hearing loss caused by a C12258A mutation in the mitochondrial MTTS2 gene.

Family ZMK is a large Irish kindred that segregates progressive sensorineural hearing loss and retinitis pigmentosa. The symptoms in the family are almost identical to those observed in Usher syndrome type III. Unlike that in Usher syndrome type III, the inheritance pattern in this family is compatible with dominant, X-linked dominant, or maternal inheritance. Prior linkage studies had resulted in exclusion of most candidate loci and >90% of the genome. A tentative location for a causative nuclear gene had been established on 9q; however, it is notable that no markers were found at zero recombination with respect to the disease gene. The marked variability in symptoms, together with the observation of subclinical muscle abnormalities in a single muscle biopsy, stimulated sequencing of the entire mtDNA in affected and unaffected individuals. This revealed a number of previously reported polymorphisms and/or silent substitutions. However, a C-->A transversion at position 12258 in the gene encoding the second mitochondrial serine tRNA, MTTS2, was heteroplasmic and was found in family members only. This sequence change was not present in 270 normal individuals from the same ethnic background. The consensus C at this position is highly conserved and is present in species as divergent from Homo sapiens as vulture and platypus. The mutation probably disrupts the amino acid-acceptor stem of the tRNA molecule, affecting aminoacylation of the tRNA and thereby reducing the efficiency and accuracy of mitochondrial translation. In summary, the data presented provide substantial evidence that the C12258A mtDNA mutation is causative of the disease phenotype in family ZMK.

A novel Asp380Ala mutation in the GLC1A/myocilin gene in a family with juvenile onset primary open angle glaucoma.

Glaucoma describes a clinically and genetically heterogeneous group of diseases that result in optic neuropathy and progressive loss of visual fields. A gene for juvenile onset primary open angle glaucoma JOAG) has recently been mapped to 1q21-31. Mutations in the trabecular meshwork induced glucocorticoid response gene (TIGR, also known as myocilin or the GLC1A locus) have been found to cause both juvenile and later onset primary open angle glaucoma. Family TCD-POAG1 is a Spanish kindred, which segregates JOAG in an autosomal dominant fashion. This family was found to be linked to the previously identified GLC1A locus on chromosome 1q. Direct sequencing of the TIGR/myocilin gene showed a heterozygous A to C transition in codon 380, resulting in the substitution of alanine for aspartic acid (Asp380Ala). This substitution created a StyI restriction site, which segregated with the JOAG phenotype and permitted rapid screening of all members of the family. This restriction site was not present in 60 controls.

A synthetic peptide ligase.

The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.

Strategems in vitro for gene therapies directed to dominant mutations.

A major difficulty associated with the design of gene therapies for autosomal dominant diseases is the immense intragenic heterogeneity often encountered in such conditions. In order to overcome such difficulties we have designed, and evaluated in vitro, three strategies which avoid a requirement to target individual mutations for genetic suppression. In the first, normal and mutant alleles are suppressed by targeting sequences in transcribed but untranslated regions of transcript (UTRs), enabling introduction of a replacement gene with the correct coding sequencing but altered UTRs to prevent suppression. A second approach involves suppression in coding sequence and concurrent introduction of a replacement gene by exploiting the degeneracy of the genetic code. A third strategy utilises intragenic polymorphism to suppress the disease allele specifically, the advantage being that a proportion of patients with different disease mutations have the same polymorphism. These approaches provide a wider choice of target sequence than those directed to single disease mutations and are appropriate for many mutations within a given gene. General methods for suppression may be directed towards the primary defect or a secondary effect associated with the disease process, such as apoptosis. Three general methods targeting the primary defect which circumvent problems of allelic genetic heterogeneity are explored in vitro using hammerhead ribozymes designed to target transcripts from the rhodopsin, peripherin and collagen 1A1 and 1A2 genes, extensive genetic heterogeneity being a feature of associated disease pathologies.

"Activation" of vitamin D by the liver.

Isolation of the liver from the circulation of rats eliminates almost completely their ability to convert [1,2]-(3)H vitamin D(3) into its biologically active metabolite, 25-hydroxycholecalciferol, as well as certain other metabolites. It is concluded that the liver is the major if not the only physiologic site of hydroxylation of vitamin D(3) (cholecalciferol) into 25-hydroxycholecalciferol. The osteodystrophy and the higher requirements for vitamin D observed in hepatic insufficiencies may be due to an inability of the liver to transform vitamin D into its metabolically active form.