Positional cloning of the gene for X-linked retinitis pigmentosa 2.

X-linked retinitis pigmentosa (XLRP) results from mutations in at least two different loci, designated RP2 and RP3, located at Xp11.3 and Xp21.1, respectively. The RP3 gene was recently isolated by positional cloning, whereas the RP2 locus was mapped genetically to a 5-cM interval. We have screened this region for genomic rearrangements by the YAC representation hybridization (YRH) technique and detected a LINE1 (L1) insertion in one XLRP patient. The L1 retrotransposition occurred in an intron of a novel gene that consisted of five exons and encoded a polypeptide of 350 amino acids. Subsequently, nonsense, missense and frameshift mutations, as well as two small deletions, were identified in six additional patients. The predicted gene product shows homology with human cofactor C, a protein involved in the ultimate step of beta-tubulin folding. Our data provide evidence that mutations in this gene, designated RP2, are responsible for progressive retinal degeneration.

The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein.

X-linked congenital stationary night blindness (XLCSNB) is characterized by impaired scotopic vision with associated ocular symptoms such as myopia, hyperopia, nystagmus and reduced visual acuity. Genetic mapping in families with XLCSNB revealed two different loci on the proximal short arm of the X chromosome. These two genetic subtypes can be distinguished on the basis of electroretinogram (ERG) responses and psychophysical testing as a complete (CSNB1) and an incomplete (CSNB2) form. The CSNB1 locus has been mapped to a 5-cM linkage interval in Xp11.4 (refs 2,5-7). Here we construct and analyse a contig between the markers DXS993 and DXS228, leading to the identification of a new gene mutated in CSNB1 patients. It is partially deleted in 3 families and mutation analysis in a further 21 families detected another 13 different mutations. This gene, designated NYX, encodes a protein of 481 amino acids (nyctalopin) and is expressed at low levels in tissues including retina, brain, testis and muscle. The predicted polypeptide is a glycosylphosphatidylinositol (GPI)-anchored extracellular protein with 11 typical and 2 cysteine-rich, leucine-rich repeats (LRRs). This motif is important for protein-protein interactions and members of the LRR superfamily are involved in cell adhesion and axon guidance. Future functional analysis of nyctalopin might therefore give insight into the fine-regulation of cell-cell contacts in the retina.

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death.

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.

Comparative process analysis of fullerene production by the arc and the radio-frequency discharge methods.

In this work, comparative analysis of processes in carbon arc and radio frequency (RF) plasma during fullerene synthesis has been presented. The kinetic model of fullerene formation developed earlier has been verified in both types of plasma reactors. The fullerene yield depended on carbon concentration, velocity of plasma flame and rotational temperature of C2 radicals predominantly. When mean rotational temperature of C2 radicals was 3000 K, the fullerene yield was the highest regardless of the type of used reactor. The zone of fullerene formation is larger significantly in RF plasma reactor compared to arc reactor.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase.

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.

Crystallization and preliminary X-ray diffraction studies of a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina.

Crystals of a glutathione S-transferase from the Australian sheep blowfly Lucilia cuprina have been grown from ammonium sulphate by the hanging drop vapour diffusion method. Successful crystallization required the presence of the inhibitor S-hexylglutathione. The crystals belong to the tetragonal space group P4(1)22 (or P4(3)22) with cell dimensions of a = b = 88.1 A and c = 66.9 A. They contain one monomer in the asymmetric unit and diffract beyond 2.8 A resolution.

Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina.

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIalpha isoform is prominent in the cerebellum and medulla, whereas the cGKIbeta isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase.

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Cleaved antitrypsin polymers at atomic resolution.

Alpha1-antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of alpha1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that alpha1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the beta-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a beta-strand linked polymer form of alpha1-antitrypsin: the crystal structure of a cleaved alpha1-antitrypsin polymer.

Crystallization and preliminary X-ray analysis of a thiol-activated cytolysin.

We present the first reported crystallization of a member of the thiol-activated family of protein toxins. Perfringolysin O, a virulence factor of Clostridium perfringens, has been crystallized in two different forms by the hanging drop vapor diffusion method. In one form the toxin crystallizes with PEG 20000 in the orthorhombic space group C222(1) with cell dimensions of a = 47.8 A, b = 182.0 A and c = 175.5 A and the crystals diffract to beyond 2.5 A resolution. In the second form the toxin crystallizes in a large variety of organic solvents including malt whisky. This crystal form belongs to the orthorhombic space group P222(1) with unit cell dimensions a = 47.1 A, b = 166.1 A and c = 214.0 A and with diffraction observed to 2.4 A resolution.

Conjunctival mucoepidermoid carcinoma in a young HIV-infected man.

PURPOSE: To report a case of conjunctival mucoepidermoid carcinoma occurring in a long-standing pterygium in a 33-year-old Cambodian man infected with the human immunodeficiency virus (HIV). METHODS: Review of clinical history and histopathologic findings. RESULTS: A pterygium that was present for 8 years suddenly became highly inflamed and underwent rapid growth. After the initial diagnostic conjunctival and corneal biopsy showed mucoepidermoid carcinoma, subsequent additional deep excisions of the adjacent sclera and cornea were necessary to completely excise the tumor. Cytokeratin and mucicarmine stains were used to confirm the pathologic diagnosis of mucoepidermoid carcinoma. CONCLUSIONS: Unique features of this case include the extremely young age of the patient (perhaps rendered susceptible by his HIV infection), the tumor masquerading as a pterygium, and the use of a hybrid lamellar and full-thickness corneoscleral resection requiring a complementary graft. Seventeen months after the resection, the patient is free of tumor; this was histopathologically confirmed with multiple random conjunctival biopsies.

Genotype-phenotype correlation in X-linked retinitis pigmentosa 2 (RP2).

PURPOSE: To identify possible correlations between the putative mutations and the clinical characteristics in X-linked retinitis pigmentosa, RP2. DESIGN: A retrospective, descriptive clinical study. MATERIAL: The ophthalmological files on affected persons from three Danish families with identified pathogenic mutations in the RP2 gene. RESULTS: Mutation analysis in 14 Danish families with X-linked retinitis pigmentosa revealed disease-associated sequence alterations in eight of them. Five mutations were detected in the RP3 gene (RPGR) and three in the RP2 gene. Genotype-phenotype comparison in the three RP2 families revealed striking interfamilial phenotypic differences. Severe phenotypes were associated with a null mutation Gln26stop and a missense mutation Arg118His. These families differed mutually with respect to retinal appearance. Affected carriers had a delayed onset by three decades. Tapetal reflexes were not observed in the carriers. An in-frame deletion DeltaSer6 was associated with a milder phenotype. CONCLUSIONS: Interfamilial differences in RP2 phenotype might be related to the type and location of the mutational event. Due to a considerable overlap between RP2 and RP3 phenotypes, the genotype cannot safely be deduced from conventional clinical examination methods.

Intrafamilial variability of the ocular phenotype in a Polish family with a missense mutation (A63D) in the Norrie disease gene.

PURPOSE: To describe the phenotypic variability in a Polish Norrie disease (ND) family associated with the missense mutation A63D. METHODS: A patient with spared vision from a Polish ND family underwent detailed ophthalmological examinations including slit-lamp biomicroscopy, ultrasound (USG), angiography, Goldmann kinetic visual field, and electroretinography (ERG). Mutation screening was carried out using the single-strand conformation polymorphism (SSCP) technique and subsequent DNA sequencing of the coding part of the ND gene. RESULTS: A mutation was detected (exon 3, A63D) in a large Polish family with 12 affected males, all but one presenting with classical ND symptoms. In one male, partially preserved vision was observed up to 40 years of age (distance acuity of the right eye 1/50 and left eye 2/50). Slit-lamp examination revealed remnants of a persistent primary vitreous and hyaloid artery. Upon angiography, the retina was vascularized within the posterior pole but not in the periphery. The ERG revealed pathological changes characteristic for chorioretinal degenerations. CONCLUSION: Within one family, individuals with identical sequence alterations in the ND gene can show remarkable phenotypic variability of the ocular symptoms. These findings indicate the involvement of additional factors (epigenetic or genetic) in ocular pathogenesis of ND.

Astigmatic decay following small incision, self-sealing cataract surgery.

A series of 22 consecutive patients had phacoemulsification using a small (3.5 to 4.0 mm), self-sealing incision. Preoperative keratometric analysis was performed using the EyeSys photokeratoscope. Results of this analysis were compared with keratometric data obtained at one week and at one month postoperatively. These comparisons were evaluated for surgery-induced cylinder and astigmatic decay at the 3, 5, and 7 mm corneal zones. At one week postoperatively, there was only mild against-the-rule change in cylinder at each corneal zone, and these changes showed minimal decay at the one month follow-up visit.

Astigmatic decay following small incision, self-sealing cataract surgery: one-year follow-up.

Twenty patients who had phacoemulsification using a small, self-sealing incision were evaluated one year after surgery for astigmatic changes. Keratometric analysis was performed using the EyeSys photokeratoscope, and data for the 3 mm corneal zone for each patient were recorded. Results were compared with those reported for the one-week and one-month postoperative periods. From one month to one year, a minimal amount of additional against-the-rule change in cylinder occurred. Because the range of changes was broad, it was difficult to predict the direction of astigmatic change (i.e., against the rule versus with the rule) that would occur over time for a given patient.

Extrusion of abnormal endothelium into the posterior corneal stroma in a patient with posterior polymorphous dystrophy.

PURPOSE: To report the presence of abnormal endothelium that extruded into the posterior corneal stroma in a patient with posterior polymorphous dystrophy. METHODS: The corneal button of a man who underwent penetrating keratoplasty for posterior polymorphous dystrophy was examined by light and electron microscopy. Immunoperoxidase staining for cytokeratins, vimentin, and the endothelial antigen recognized by monoclonal antibody 2B4.14.1 antigen was performed. Two-color immunofluorescence staining for simultaneous detection of cytokeratins and 2B4.14.1 antigen was also done. RESULTS: Much of the endothelium had characteristic features of epithelium-like cells, and abnormalities in Descemet's membrane were present. Curious oval and slit-like spaces in the posterior stroma were lined by epithelium-like endothelial cells and were continuous with the anterior chamber through defects in Descemet's membrane. CONCLUSION: These abnormalities in the posterior stroma have not previously been described in histopathologic reports of posterior polymorphous corneal dystrophy and are likely an unusual variation in the spectrum of this hereditary disorder.

A new experimental setup designed for the investigation of irradiation of nanosystems in the gas phase: a high intensity mass-and-energy selected cluster beam.

DIAM (Dispositif d'Irradiation d'Agrégats Moléculaires) is a new experimental setup devoted to investigate processes induced by irradiation at the nanoscale. The DIAM apparatus is based on a combination of techniques including a particle beam from high-energy physics, a cluster source from molecular and cluster physics, and mass spectrometry form analytical sciences. In this paper, we will describe the first part of the DIAM apparatus that consists of an ExB double spectrometer connected to a cluster ion source based on a continuous supersonic expansion in the presence of ionizing electrons. This setup produces high intensities of energy-and-mass selected molecular cluster ion beams (1000 s of counts s(-1)). The performance of the instrument will be shown through measurements of 6-8 keV beams of protonated water clusters, (H(2)O)(n)H(+) (n = 0-21) and mixed protonated (or deprotonated) water-pyridine cluster ions: PyrH(+)(H(2)O)(n) (n = 0-15), Pyr(2)H(+) (H(2)O)(n) (n = 0-9), and (Pyr-H)(+) (H(2)O).

Identification and characterization of a misfolded monomeric serpin formed at physiological temperature.

The native serpin state is kinetically trapped. However, under mildly destabilizing conditions, the conformational landscape changes, and a number of nonnative conformations with increased stability can be readily formed. The ability to undergo structural change is due to intrinsic strain within the serpin's tertiary fold, which is utilized for proteinase inhibition but renders the protein susceptible to aberrant folding and self-association. The relationship between these various conformations is poorly understood. Antichymotrypsin (ACT) is an inhibitory serpin that readily forms a number of inactive conformations, induced via either environmental stress or interaction with proteinases. Here we have used a variety of biophysical and structural techniques to characterize the relationship between some of these conformations. Incubation of ACT at physiological temperature results in the formation of a range of conformations, including both polymer and misfolded monomer. The ability to populate these nonnative states and the native conformation reflects an energy landscape that is very sensitive to the solution conditions. X-ray crystallography reveals that the misfolded monomeric conformation is in the delta conformation. Further polymerization and seeding experiments show that the delta conformation is an end point in the misfolding pathway of ACT and not an on-pathway intermediate formed during polymerization. The observation that ACT readily forms this inactive conformation at physiological temperature and pH suggests that it may have a role in both health and disease.