ADVIRC is caused by distinct mutations in BEST1 that alter pre-mRNA splicing.

Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. It has been shown previously that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. This study describes a novel ADVIRC mutation and show that it disrupts an exonic splice enhancer (ESE) site, altering the binding of a splicing-associated SR protein. As with previous ADVIRC mutations, the novel c.704T-->C mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707G-->A) either weakened or abolished splicing in an ESE-dependent splice assay compared with a nearby exon 6 mutation associated with Best disease (c.703G-->C). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. Although SC35, SRp40 and SRp55 proteins all bound to the wild-type and mutated sequences with similar intensities, there was increased binding of ASF/SF2 to the two ADVIRC-mutated sequences compared with the wild-type or Best disease-mutated sequences. The exon skipping seen for these two exon 6 ADVIRC mutations and their affinity for ASF/SF2 suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site.

Anterior sacral meningocele: management in gynecological practice.

We describe the case of a young woman with anterior sacral meningocele (ASM), initially identified during a routine ultrasound examination and subsequently diagnosed using magnetic resonance imaging (MRI). ASM is a rare disorder characterized by uni- or multilocular extensions of the meninges from the sacral spinal canal to the retroperitoneal presacral space. Common symptoms include lower back and pelvic pain, constipation, difficulties in defecation, dysmenorrhea and dyspareunia, and urinary incontinence, retention or urgency. Perineal and lower-extremity paresthesias may present when nerve roots are affected. Despite its more posterior location, ASM can mimic an ovarian cyst or other adnexal cystic mass, and in the obstetric patient can present a mechanical obstacle to delivery with a risk of rupture and infection during labor and delivery. Although it is a rare condition, we feel that awareness of the etiology, presentation and imaging characteristics of ASM is of importance and have therefore carried out a review of the literature, taking into account case findings and the obstetric and gynecological management of this disorder.

Intrafamilial phenotypic variability in families with RDS mutations: exclusion of ROM1 as a genetic modifier for those with retinitis pigmentosa.

OBJECTIVES: To identify suspected RDS mutations in families in which different people have been identified with either generalised retinal dystrophy or macular dystrophy. METHODS: Two families with a retinal dystrophy were extensively phenotyped and blood was taken for mutation analysis of the RDS (all) and ROM1 (retinitis pigmentosa patients only) genes. RESULTS: A novel p.Trp94X mutation in RDS was found in all three affected members of a two-generation family that was associated with retinitis pigmentosa in the son, pattern dystrophy in the daughter and fundus flavimaculatus in the mother. In the second family, the proband with retinitis pigmentosa carried a p.Arg220Trp mutation. The mother, who was unavailable for mutation screening, had adult vitelliform macular dystrophy. No ROM1 mutations were found in those with retinitis pigmentosa in either family. CONCLUSION: Mutations in RDS can be associated with an intrafamilial variation in retinal disease. The phenotypes range from Stargardt-like macular dystrophy to classic retinitis pigmentosa. CLINICAL RELEVANCE: Intrafamilial phenotypic variation may be due to the presence of environmental or genetic modifying factors. The presence of a modifying-sequence change in the coding region of ROM1 for two people with retinitis pigmentosa from two families with intrafamilial variation in RDS mutation phenotype has been excluded in this study.

RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin.

The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGR(ORF15)-NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGR(ORF15) fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGR(ORF15) and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGR(ORF15) from cultured cells and (iv) co-localization of NPM and RPGR(ORF15) at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.

[Mechanisms of the stagnation of dilatation in the active phase of labor]. / Mécanismes des stagnations de la dilatation en phase active du travail.

OBJECTIVE: Our study was designed to explain determinants of nonprogressive labor in nulliparous patients. STUDY DESIGN: One hundred consecutive nulliparous patients have got a cesarean section for active-phase arrest of labor after two hours of active management. Intrauterine pressure was monitored for all of them and a X-ray pelvimetry was done after surgery. RESULTS: Eighty-three percent of patients showed X-ray data considered as normal; hypotonic labor was found in 50% of cases and occiput posterior position in 60%. CONCLUSIONS: Our results suggest that occiput position and functional dystocia are more common in case of nonprogressive labor than abnormal measurements of the obstetrical pelvis.

Identification of a 5' splice site mutation in the RPGR gene in a family with X-linked retinitis pigmentosa (RP3).

We have identified a novel RPGR gene mutation in a large Dutch family with X-linked retinitis pigmentosa (RP3). In affected members, a G-->T transversion was found at position +1 of the 5' splice site of intron 5 of the RPGR (retinitis pigmentosa GTPase regulator) gene. Analysis of this mutation at the RNA level showed cryptic splicing upstream of the mutation in exon 5 leading to a frameshift and downstream termination codon. Identification of the causative mutation in this family has facilitated the detection of females at risk of having an affected son.

The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase.

Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR). The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTP-binding protein Ran. In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDEdelta) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR392). The interaction of RPGR with PDEdelta was confirmed by pull-down assays and plasmon surface resonance. The binding affinity was determined to be 90 nM. Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system. All missense mutations showed reduced interaction with PDEdelta. A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDEdelta. PDEdelta is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family. These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.

Epidemiology of lameness in dairy cattle: the influence of cubicles and indoor and outdoor walking surfaces.

A survey of cubicles and indoor and outdoor walking surfaces on 37 farms served by four veterinary practices in Somerset, Cheshire, Wirral and west. Wales was carried out in 1989 to 1991. A study of the space requirements of Friesian/Holstein cows at pasture showed that they required approximately 240 cm x 120 cm lying space and a further 60 cm lunging space for rising. By these standards, 87 per cent of the cubicles were too short and 50 per cent were too wide or too narrow. Over 1500 observations on cows lying down, rising and standing indicated that only 12 per cent of the cubicles permitted real freedom of movement; 91 per cent of top partition rails were judged to be too low and 70 per cent of bottom rails too low or too high. In addition, the kerb was very high in 76 per cent of the cubicles. As a result, 10 per cent of cows appeared moderately or severely restricted when lying down, 33 per cent when rising and 55 per cent when standing. Over 2000 cubicle beds were also studied; 75 per cent had a concrete base and of those, 63 per cent were judged to have too little bedding and 11 per cent next to none. Higher incidences and prevalences of lameness were associated with limited borrowing space (P < 0.01) low bottom rails (P < 0.05), high kerbs (P < 0.05) and inadequate bedding (P < 0.01). Of 3190 walking surfaces, only 25 per cent were classified as satisfactory in the first winter and 34 per cent in the second. In general, surfaces in silage bays were too rough and those in other sites were too smooth. The farms with the smoothest indoor walking surfaces had a significantly higher incidence of lameness (P < 0.01). Of 3335 outdoor walking surfaces only 25 per cent were classified as satisfactory, and 70 per cent were too rough. The incidence of lameness was not significantly related to these findings.

Incidence and prevalence of lameness in dairy cattle.

A survey was made of 37 dairy farms in Wirral, mid-Cheshire, mid-Somerset and Dyfed, Wales, to assess the incidence and prevalence of lameness in the cows between May 1989 and September 1991. The incidence was obtained from records made whenever a cow was examined for lameness or received preventive foot-trimming. The mean annual incidence was 54.6 new cases per 100 cows with a range from 10.7 to 170.1 and the mean values during summer and winter were 22.9 and 31.7, respectively. The prevalence of lameness was measured by regular visits at which locomotion was scored on a scale of 1 to 5, and the prevalence of lameness was calculated for each visit as the proportion of cows with scores of 3 or more. The mean annual prevalence over the whole period was 20.6 per cent with a range from 2.0 to 53.9 per cent for the 37 farms. The mean prevalences during summer and winter were 18.6 and 25.0 per cent, respectively. The prevalence measured at a single visit in midsummer or midwinter was significantly correlated with the mean prevalence over the whole corresponding period and may be useful as an assessment of the extent of lameness in a herd and the efficacy of control measures. There was evidence that training farmers to recognise early cases of lameness and request veterinary treatment resulted in a marked reduction in the duration of cases of lameness.

Epidemiology of lameness in dairy cattle: description and analysis of foot lesions.

Information from 37 dairy farms, in four regions of England and Wales provided data on 8991 lesions and the preventive trimming of 4837 cows' feet. Of the total of 13,828 forms returned, veterinary surgeons treated 32 per cent and farmers or stockmen 46 per cent. Of the 8645 lesions associated with episodes of lameness, lesions in the hindlimbs accounted for 92 per cent, of which 65 per cent were in the outer claw, 20 per cent in the skin and 14 per cent in the inner claw. Sole ulcers (40 per cent) and white line lesions (29 per cent) were the predominant diseases of horn, and digital dermatitis (40 per cent) was the most common disease of the skin. Subjective assessments showed that sandcrack, penetration of the sole by foreign bodies and interdigital necrobacillosis were associated with the most severe cases of lameness. There was a significant seasonal effect in the reporting of lesions.

A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X-linked retinitis pigmentosa (RP3).

X-linked retinitis pigmentosa (xlRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population. The most common form of xlRP, RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping. Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kg of genomic DNA and is ubiquitously expressed. The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase. The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site. The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.

Interaction of cytochrome c with flavocytochrome b2.

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 [per cytochrome c reduced; Miles, C. S., Rouviere, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) muM-1 s-1 in the presence of 10 mM L-lactate. This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-transfer rate in the pre-formed complex being in excess of 1000 s-1. Inhibition of the pre-steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate. The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S. & Cambillau, C. (1993) Proteins 16, 408-422] has been tested using site-directed mutagenesis. Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere. The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency. In the steady-state, the turnover rate is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue [Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6345-6350].

Identification of a novel gene, ETX1 from Xp21.1, a candidate gene for X-linked retintis pigmentosa (RP3).

A novel gene encoding a 2.2 kilobase transcript has been isolated from the Xp21.1 region of the human X chromosome by exon amplification. The gene, called EXT1, spans 80 kilobases and contains 12 exons, at least two of which are alternatively spliced and have predicted products of 464 and 471 amino acids respectively. Conceptual translation of the open reading frames shows one product with a 30 amino acid signal peptide, which is absent from the alternative transcript, followed by three complement control protein domains, a hydrophobic region with a possible role in membrane anchorage and short 17 amino acid putative cytoplasmic carboxyl terminus. An alternative first exon contains a 39 amino acid open reading frame which is rich in serine and threonine residues and contains a potential chondroitin/dermatan sulphate attachment site. Northern analysis showed ETX1 expression within the retina and heart with lower levels in several other tissues. Since ETX1 lies within the region thought to contain the x-linked retinitis pigmentosa (xIRP) gene, RP3, it was screened for mutation within a set of 45 xIRP patients using single strand conformation analysis and/or chemical cleavage of mismatch using reverse transcription/polymerase chain reaction amplification of polyA+RNA from blood cells. Three low frequently variants (17-23Ldel, P225S, S413F) were found in both patients and controls; one of which (P225S) was found in four of 45 unrelated patient chromosomes and one of 178 control chromosomes (p <0.001). The allelic association between P225S and xIRP alleles suggests a common ancestral chromosome bearing the P225S variant and an RP3 mutation at a neighbouring locus.

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.

Mutation to glutamine of histidine 373, the catalytic base of flavocytochrome b2 (L-lactate dehydrogenase).

Flavocytochrome b2 catalyzes the two-electron oxidation of L-lactate. Reducing equivalents are transferred first to FMN then to heme b2 in the same subunit, finally to cytochrome c or a non-physiological acceptor. The enzyme's three-dimensional structure, when analyzed in the light of existing mechanistic knowledge, suggested that His 373 is the active site base which initiates the substrate chemical transformation by abstracting the lactate alpha-proton. We report here the properties of a mutant enzyme with glutamine substituted histidine at position 373. The mutated enzyme preparations show a 10(4)-fold decrease in catalytic activity. We find that most of this residual activity can be eliminated by treatments with: 1) fluoropyruvate, an affinity label for His 373; and 2) 2- hydroxy-3-butynoate, a suicide reagent which normally forms an adduct with FMN but in this case leaves the bulk of the prosthetic group intact. Furthermore, although spectral titrations do not detect any binding of oxalate, this reagent inhibits the mutant enzyme with the same kinetic behaviour as for the wild-type enzyme. We conclude that the enzyme preparations contain about 1 in 10(4) molecules of wild-type flavocytochrome b2; this is probably due to codon misreading during biosynthesis. Thus the H373Q enzyme displays at most 10(5)-fold less activity than the wild-type enzyme. We report values for the spectrally determined binding constants of sulfite, pyruvate and D-lactate for the mutant enzyme. Finally, we show that 2,6-dichlorophenol indophenol, which is a 10-fold more sensitive routine electron acceptor than ferricyanide, accepts electrons only from heme b2 and not from the flavin.

Purification and properties of a novel cytochrome: flavocytochrome c from Shewanella putrefaciens.

The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.