Ethylnitrosourea-induced mutation in mice leads to the expression of a novel protein in the eye and to dominant cataracts.

A novel ENU-induced mutation in the mouse leading to a nuclear and zonular opacity of the eye lens (Aey1) was mapped to chromosome 1 between the markers D1Mit303 and D1Mit332. On the basis of the chromosomal position, the gamma-crystallin encoding gene cluster (Cryg) and the betaA2-crystallin encoding gene Cryba2 were tested as candidate genes. An A --> T mutation destroys the start codon of the Cryge gene in the mutants; this mutation was confirmed by the absence of a restriction site for NcoI in the corresponding genomic fragment of homozygous mutants. The next in-frame start codon is 129 bp downstream; this predicted truncated gammaE-crystallin consists of 131 amino acids, resulting in a molecular mass of 14 kD. However, another open reading frame was observed just 19 bp downstream of the regular Cryge start codon, resulting in a protein of 119 amino acids and a calculated molecular weight of 13 kD. Western blot analysis using polyclonal antibodies against gamma-crystallins or the novel Aey1-specific protein demonstrated the specific expression of the Aey1 protein in the cataractous lenses only; the truncated form of the gammaE-crystallin could not be detected. Therefore, it is concluded that the novel protein destroys the sensitive cellular structure of the eye lens.

Superoxide dismutase activity and expression in human venous and arterial bypass graft vessels.

UNLABELLED: Venous bypass grafts are more prone to accelerated atherosclerosis than arterial grafts, which is partly related to increased oxidative stress and diminished nitric oxide bioavailability. In veins superoxide production is dependent primarily on nox2 NAD(P)H oxidase expression, while in arteries nox4 appears to play an important role. This may in part explain differences in susceptibility to graft failure. Net levels of oxidative stress are however determined in parallel by the production as well as by degradation of free radicals (eg. by superoxide dismutases, catalases, thioredoxins etc). The differences in superoxide dismutase (SOD) expression and activity in human bypass conduit vessels remain unclear. Accordingly, we aimed to compare SOD activity and protein levels as well as its functional effects on superoxide production in segments of human internal mammary arteries (IMA) and saphenous veins (HSV) from patients undergoing bypass graft surgery (n=24). SOD activity was assessed by inhibition of pyrogallol autoxidation, Cu-Zn SOD and Mn SOD protein levels were studied by immunoblotting. Basal superoxide release was detected by lucigenin (5 microM) enhanced chemiluminescence. Total SOD activity did not differ significantly between HSV and IMA. Similarly, no difference was observed in SOD activity in the presence of KCN (Mn-SOD). Human bypass conduit vessels show amounts of Cu-Zn SOD or Mn-SOD protein levels. In both HSV and IMA segments superoxide production was more than doubled in the presence of SOD inhibitor-DETC. CONCLUSIONS: These studies suggest that the differences in oxidative stress between human arteries and veins are unlikely to be caused by SOD activity. However SOD plays and important role in amelioration of oxidative stress in both types of vessels.

Sequence analysis of the beta B2-crystallin cDNA of hamster containing a domain conserved among vertebrates.

The cDNA sequence of the beta B2-cry was determined from hamster (Mesocricetus auratus) and compared to the corresponding genes of bovine, frog, chicken, human, mouse and rat. Multispecies comparison demonstrated high homology between the hamster, rat and mouse gene, but larger distances to man, bovine, chicken and frog. There is striking identity within a strech of 36 deduced amino acids (aa) between the Greek key motif 3 and part of motif 4. This 36-aa domain contains a putative phosphorylation site for protein kinase C and is highly conserved among all known basic beta B-Cry; however, it can neither be detected in the acidic beta A-nor in the gamma-Cry.

Characterization of a 1-bp deletion in the gammaE-crystallin gene leading to a nuclear and zonular cataract in the mouse.

PURPOSE: A previous study had found a mouse mutant to have bilateral nuclear cataract with zonular opacity after paternal irradiation with gamma-rays. The mutation was then demonstrated to be allelic with the Cat2 group of dominant cataract mutations and was referred to as Cat2(nz) in a later study. Because several members of this group have been confirmed as mutations in the gene cluster coding for gamma-crystallins (CRYG:), these genes were now tested as candidates for Cat2(nz). METHODS: All six gamma-crystallin-encoding genes were amplified by polymerase chain reaction (PCR) from cDNA or genomic DNA and sequenced. An antibody against the changed protein was developed and used for Western blot analysis. The mutant was also characterized morphologically. RESULTS: A 1-bp deletion in exon 2 of the gammaE-crystallin-encoding gene CRYGE: was causative of the cataract phenotype. This particular mutation is therefore referred to as CRYGE:(nz). The predicted frameshift after codon 29 led to a changed amino acid sequence of 96 amino acids. The altered 13-kDa protein was expressed in the eye lens as demonstrated by Western blot analysis. Cataracts became visible at day 18.5 of embryonic development and reached the final phenotype at 2 weeks after birth. CONCLUSIONS: The CRYGE:(nz) is the sixth mutation in the mouse that has been reported so far to affect the CRYG: gene cluster, which demonstrates its importance for lens transparency.

Characterization of a new, dominant V124E mutation in the mouse alphaA-crystallin-encoding gene.

PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screening, mice were tested for the occurrence of dominant cataracts. The purpose of the study was morphologic description, mapping of the mutant gene, and characterization of the underlying molecular lesion in a particular mutant, Aey7. METHODS: Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed with a set of microsatellite markers covering all autosomal chromosomes. cDNA was amplified after reverse transcription of lens mRNA. For PCR, cDNA or genomic DNA was used as a template. RESULTS: Nuclear opacity and posterior suture anomaly were visible at eye opening and progressed to a nuclear and zonular cataract at 2 months of age. The opacity as well as the microphthalmia was more pronounced in the homozygotes than in the heterozygotes. The mutation was mapped to chromosome 17 between the markers D17Mit133 and D17Mit180. This position made the alphaA-crystallin-encoding gene (Cryaa) an excellent candidate gene. Sequence analysis revealed a mutation of a T to an A at position 371 in the Cryaa cDNA. The mutation was confirmed by an additional MnlI restriction site in the genomic DNA of homozygous mutants leading to replacement of Val with Glu at codon 124 affecting the C-terminal region of the alphaA-crystallin. CONCLUSIONS: The Aey7 mutant represents the first dominant mouse cataract mutation affecting the Cryaa gene. The mutation leads to progressive opacification of the lens. Compared with the beta- and gamma-crystallin-encoding genes, mutations in the alpha-crystallin-encoding genes are rare.

Aey2, a new mutation in the betaB2-crystallin-encoding gene of the mouse.

PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was found that caused progressive opacity and was referred to as Aey2. The purpose of the study was to provide a morphologic description, to map the mutant gene, and to characterize the underlying molecular lesion. METHODS: Isolated lenses were photographed, and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed using a set of microsatellite markers covering all autosomal chromosomes. cDNA from candidate genes was amplified after reverse transcription of lens mRNA. RESULTS: The cortical opacification visible at eye opening progressed to an anterior suture cataract and reached its final phenotype as total opacity at 8 weeks of age. There was no obvious difference between heterozygous and homozygous mutants. The mutation was mapped to chromosome 5 proximal to the marker D5Mit138 (8.7 +/- 4.2 centimorgan [cM]) and distal to D5Mit15 (12.8 +/- 5.4 cM). No recombinations were observed to the markers D5Mit10 and D5Mit25. This position makes the genes within the betaA4/betaB-crystallin gene cluster excellent candidate genes. Sequence analysis revealed a mutation of T-->A at position 553 in the Crybb2 gene, leading to an exchange of Val for GLU: It affects the same region of the Crybb2 gene as in the Philly mouse. Correspondingly, the loss of the fourth Greek key motif is to be expected. CONCLUSIONS: The Aey2 mutant represents the second allele of Crybb2 in mice. Because an increasing number of beta- and gamma-crystallin mutations have been reported, a detailed phenotype-genotype correlation will allow a clearer functional understanding of beta- and gamma-crystallins.

Prevalence and characterization of Helicobacter pylori (H. pylori) infection and colonization in dentists.

H. pylori is an important factor in the pathogenesis of numerous diseases including gastro-intestinal, metabolic and vascular disorders. Therefore, identification of individuals at risk of this infection remains of critical importance. Dentists and dental professionals may be at increased risk due to the contact with oral cavity of patients with the presence of H. pylori in the oral cavity where it may serve as reservoir for gastric infections and participate in the pathogenesis oral mucosal lesions and ulceration. However, evidence regarding the occurrence of H. pylori infections and colonization in dentists is conflicting, but has been based mainly on serological studies, which carry significant limitations. Therefore, we attempted to characterize H. pylori infection in practising dentists in relation to the duration of their work as dental professionals. Moreover, apart from seropositivity, which was used by majority of previous studies, we have performed urea-breath test (UBT), which has been shown to represent active H. pylori infection in stomach as well as the H. pylori culture from the oral cavity. We found that while the occurrence of either gastric or oral H. pylori in dentists is not greater than in general population, it seems that in male dentists there is a greater risk of gastric H. pylori infection. Moreover, we found a relationship between the length of dentist occupation with the presence of H. pylori in gingival sulcus. In conclusion, while overall occurrence of H. pylori in dentists did not differ from that reported for stomach or oral cavity in general population, there was an increased occurrence of H. pylori in male dentists and the presence of this germ in the oral cavity appears to be related to the length of professional exposure.

Cat3vl and Cat3vao cataract mutations on mouse chromosome 10: phenotypic characterization, linkage studies and analysis of candidate genes.

Cat3vl and Cat3vao are two allelic, dominant cataract mutations that arose independently in the F1 generation after gamma-irradiation of male mice. The cataracts are already present at birth. Examination of the eyes with a slit lamp revealed completely vacuolated lenses in Cat3vl mutants and anteriorly located opacity in Cat3vao mutants. The appearance of the opacities does not differ between the individuals or between heterozygotes and homozygotes. Penetrance of the mutations is complete. Viability and fertility of the mutants are normal except in the case of the Cat3vl homozygotes. Cat3vao was assigned to the distal part of mouse chromosome 10, 3.2 +/- 0.9 cM away from the visible marker Steel (SlgbH). Using polymorphic markers the following locus order was found: D10Mit230-(0.2 +/- 0.1 cM)-Cat3vao-(2.5 +/- 0.6 cM)-D10Mit70. No recombinants were found between Cat3vao and the markers D10Mit4l and D10Mit95 among 921 offspring. The results exclude allelism of Cat3vao with CatLop or To2, which also map to chromosome 10. Candidate genes were tested by examination of their expression in the eye of newborn mice and by analysis of cDNA sequences. So far, negative results have been obtained for the genes encoding the proteoglycans lumican and decorin, the nuclear orphan receptor Tr2-11 and the transcription factor Elk3. Based on syntenic homology of the Cat3 region to the human chromosome 12q, the Cat3 mutants are discussed as mouse models for cornea plana congenita in man. The recovery of the Cat3 mutations demonstrates the importance of the corresponding locus for proper eye development.

Reduced levels of gamma-crystallin transcripts during embryonic development of murine Cat2nop mutant lenses.

BACKGROUND: From previous experiments it is known that the murine dominant cataract mutants carrying the gene Cat2 have a decreased content of gamma-crystallin-specific transcripts in the juvenile lens, when the cataract is completely expressed. Moreover, the mutant locus has been mapped recently to chromosome 1, closely linked to the gamma E-crystallin gene (map distance 0.3 +/- 0.3 cM). In the present paper we describe the phenotypic changes and the gamma-crystallin expression in embryonic lenses of the Cat2nop mutants as an example for the Cat2 allelic series. METHODS: The technique of in situ hybridization was applied using a probe from the murine gamma D-crystallin gene, and, for control, from the murine alpha A-crystallin gene. Simultaneously, a series of lens sections was examined histologically. RESULTS: The presence of gamma-crystallin mRNA was demonstrated from embryonic day 13.5 (E13.5) onward, but in the mutants to a lower extent than in the wild-type lenses. However, the first morphological abnormality in the mutant lenses was observed as swelling of lens fibers at day E15.5. Progressive degeneration of the lens core followed, leading to a cataracta immatura. CONCLUSION: The reduced level of gamma-crystallin transcripts is the first alteration observable during the embryonic development of the Cat2 mutant lenses: it precedes the morphological changes. This result represents an additional line of argument that the gamma-crystallin genes may be the target of the mutation in the Cat2 mice.

Close linkage of the dominant cataract mutations (Cat-2) with Idh-1 and cryge on mouse chromosome 1.

The murine dominant gene Cat-2 was located on chromosome 1 between the loci of fuzzy and leaden. Subsequent linkage analysis revealed one recombinant between Cat-2t and isocitrate dehydrogenase-1, and one between Cat-2t and gamma E-crystallin among 338 offspring in three-point backcrosses. The resulting genetic distance between the loci is 0.3 +/- 0.3 cM. The very close linkage between the Cat-2 and the gamma-crystallin gene cluster together with the finding of reduced gamma-crystallin transcripts in mutant lenses suggest strongly that the gamma-crystallin genes may be candidate genes for the Cat-2 mutations.

Aphakia (ak), a mouse mutation affecting early eye development: fine mapping, consideration of candidate genes and altered Pax6 and Six3 gene expression pattern.

The homozygous mouse mutant aphakia (ak) has been characterized by bilaterally aphakic eyes without a pupil [Varnum DS, Stevens, LC (1968): J Hered 59:147-150]. The mutation was mapped to chromosome 19 [Varnum DS, Stevens, LC (1975): Mouse News Lett 53:35]. Our linkage studies yielded a precise localization of the ak gene 0.6 +/- 0.3 cM proximal to the microsatellite marker D19Mit10 and 0.7 +/- 0.4 cM distal to D19Mit4 and D19Mit91. No recombination was found with the marker D19Mit9 among 418 backcross offspring tested. The developmental control gene Pax2 mapped 11.0 +/- 3.5 cM proximal to ak and is excluded as a candidate gene. Sequence analysis of Fgf8 and Chuk1, which are localized close to the marker D19Mit10, detected no mutations in the ak/ak mutants. Histological analysis of homozygous mutants suggested the arrest of lens development at the lens stalk stage, a transient morphological structure during the formation of the lens vesicle. In the lens remnants, Pax6 and Six3 are expressed, whereas in the persisting lens stalk only Pax6 was detected. The expression pattern of Pax2 appeared normal; Cryaa expression could not be detected. As a consequence of the arrested lens development, other ocular tissues that require for their development information from the intact lens, such as iris, ciliary muscle, retina, and vitreous body, are absent or formed abnormally.

Characterization of a mutation in the lens-specific MP70 encoding gene of the mouse leading to a dominant cataract.

During an ethylnitrosourea mutagenesis screen, Aey5, a new mouse mutation exhibiting an autosomal dominant congenital cataract was isolated. The cataractous phenotype is visible at the eye opening and progresses to a nuclear and zonular cataract at 2 months of age with no difference in onset or severity between heterozygous and homozygous mutants. Histological analysis revealed that fiber cell differentiation continues at the lens bow region, but the cell nuclei do not degrade normally and remain in the deeper cortex. Further, the lens nucleus has clefts of various sizes while the remainder of the eye was morphologically normal. The mutation was mapped to chromosome 3 between the markers D3Mit101 and D3Mit77 near the connexin encoding genes Gja5 and Gja8. Sequence analysis revealed no differences in the Gja5 gene, but identified a T-->C mutation at position 191 in the Gja8 gene, which was confirmed by an additional Mva 12691 restriction site in the genomic DNA of homozygous mutants. This mutation results in Val-->Ala substitution at codon 64 of connexin50 (Cx50) also known as lens membrane protein 70 (MP70). Aey5 represents the second dominant mouse cataract mutant affecting Cx50, a membrane protein preferentially expressed in the lens. Since both mutations affect similar regions in the first extracellular domain this region appears to be critically important for its function in lens transparency.

Mutation in the betaA3/A1-crystallin encoding gene Cryba1 causes a dominant cataract in the mouse.

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 +/- 4.4 cM) and proximal to D11Mit36 (2.3 +/- 2.3 cM). This position makes the betaA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected betaA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1(po1). This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene.

Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes.

A number of murine cataract mutations have been localized to chromosome 1 close to the gamma-crystallin gene cluster (Cryg) (Everett et al., 1994, Genomics 20: 429-434; Löster et al., 1994, Genomics 23: 240-242). Based on the size of the mapping or allelism tests they have not been shown to be genetically distinct and have been assigned to locus symbol Cat2. Here we assign three mutations to the respective gamma-crystallin gene. Using a systematic candidate gene approach to analyze the entire Cryg cluster, an A-->G transition was found in exon 2 of Cryga for the ENU-436 mutation and is designated Cryga1Neu. The mutant allele Crygbnop (formerly Cat2(nop)) is caused by a replacement of 11 bp by 4 bp in the third exon of Crygb, while a C-->G transversion in exon 3 of Cryge has been found for the Cryget (formerly Cat2(t)) mutation. For the mutation Cryga1Neu, an Asp-->Gly exchange is deduced, whereas the mutations Crygbnop and Cryget lead to the formation of in-frame stop codons and give rise to truncated proteins of 144 and 143 amino acids, respectively. The effects of the mutations upon gamma-crystallin structure are likely to be quite different. The Cryga1Neu mutation is expected to affect the link between Greek-key motifs 2 and 3, whereas both Crygbnop and Cryget mutations are supposed to truncate the fourth Greek-key motif. All three mutations are predicted to alter protein folding of the gamma-crystallins and result in lens cataract, but the phenotype for each is quite distinctive.