Methyl transferases induced during chemical adaptation of M. luteus.

Three peaks of methyltransferase activity specific for MNNG alkylated DNA have been identified from extracts of chemically adapted M. luteus. They are designated as TI to TIII in order to their elution from a Sephadex G-75 column. The first one of these peaks has been purified to homogeneity. TI, is an inducible, unusually salt resistant, heat labile protein which corrects O6-methylguanine in alkylated DNA by the transfer of the O6-alkyl group to a cysteine amino acid in the TI protein. There is a stoichiometric relationship between the loss of O6-methylguanine from the DNA and the production of S-methylcysteine. Partially purified TII & TIII proteins show specificity for O4-alkylthymine and methyl phosphotriesters respectively. The mode of repair by the isolated methyltransferases is similar yet there is no competition for substrate specificity. The apparent molecular weights of TI, TII & TIII proteins are 31Kd, 22Kd, and 13Kd respectively.     

An Alteration in ELMOD3, an Arl2 GTPase-Activating Protein,Is Associated with Hearing Impairment in Humans

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.     

CDH23 mutation and phenotype heterogeneity: a profile of 107 diverse families with Usher syndrome and nonsyndromic deafness

Usher syndrome type I is characterized by congenital hearing loss, retinitis pigmentosa (RP), and variable vestibular areflexia. Usher syndrome type ID, one of seven Usher syndrome type I genetic localizations, have been mapped to a chromosomal interval that overlaps with a nonsyndromic-deafness localization, DFNB12. Mutations in CDH23, a gene that encodes a putative cell-adhesion protein with multiple cadherin-like domains, are responsible for both Usher syndrome and DFNB12 nonsyndromic deafness. Specific CDH23 mutational defects have been identified that differentiate these two phenotypes. Only missense mutations of CDH23 have been observed in families with nonsyndromic deafness, whereas nonsense, frameshift, splice-site, and missense mutations have been identified in families with Usher syndrome. In the present study, a panel of 69 probands with Usher syndrome and 38 probands with recessive nonsyndromic deafness were screened for the presence of mutations in the entire coding region of CDH23, by heteroduplex, single-strand conformation polymorphism, and direct sequence analyses. A total of 36 different CDH23 mutations were detected in 45 families; 33 of these mutations were novel, including 18 missense, 3 nonsense, 5 splicing defects, 5 microdeletions, and 2 insertions. A total of seven mutations were common to more than one family. Numerous exonic and intronic polymorphisms also were detected. Results of ophthalmologic examinations of the patients with nonsyndromic deafness have found asymptomatic RP-like manifestations, indicating that missense mutations may have a subtle effect in the retina. Furthermore, patients with mutations in CDH23 display a wide range of hearing loss and RP phenotypes, differing in severity, age at onset, type, and the presence or absence of vestibular areflexia.     

Radioprotective Effect of Aminothiol PrC-210 on Irradiated Inner Ear of Guinea Pig

Radiotherapy of individuals suffering with head & neck or brain tumors subserve the risk of sensorineural hearing loss. Here, we evaluated the protective effect of Aminothiol PrC-210 (3-(methyl-amino)-2-((methylamino)methyl)propane-1-thiol) on the irradiated inner ear of guinea pigs. An intra-peritoneal or intra-tympanic dose of PrC-210 was administered prior to receiving a dose of gamma radiation (3000 cGy) to each ear. Auditory Brainstem Responses (ABRs) were recorded one week and two weeks after the radiation and compared with the sham animal group. ABR thresholds of guinea pigs that received an intra-peritoneal dose of PrC-210 were significantly better compared to the non-treated, control animals at one week post-radiation. Morphologic analysis of the inner ear revealed significant inflammation and degeneration of the spiral ganglion in the irradiated animals not treated with PrC-210. In contrast, when treated with PrC-210 the radiation effect and injury to the spiral ganglion was significantly alleviated. PrC-210 had no apparent cytotoxic effect in vivo and did not affect the morphology or count of cochlear hair cells. These findings suggest that aminothiol PrC-210 attenuated radiation-induced cochlea damage for at least one week and protected hearing.     

A Mutation in SLC24A1 Implicated in Autosomal-Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.     

Severe retinitis pigmentosa mapped to 4p15 and associated with a novel mutation in the PROM1 gene

Mutation in the PROM1 gene previously has been identified in one family with retinal degeneration for which neither ERG recordings nor detailed information about visual impairment is available. A large family with multiple individuals affected by retinal degeneration was ascertained in the Punjab province of Pakistan. The visual acuity of all affected patients in the family was severely compromised beginning in early childhood. The retinal disease in this family is a severe form of retinitis pigmentosa (RP) accompanied by macular degeneration. Fundus changes advanced with age. Choriocapillaris atrophy and posterior RPE atrophy were obvious allowing visualization of the large choroidal vessels in patients over 40 years of age. Rod and cone responses on ERG recordings were extinguished in patient’s teens. A genome-wide scan mapped the disease to a 34.7 cM region of chromosome 4p14–p16 between D4S1599 and D4S405. A maximum lod score of 3.96 with D4S403 and D4S391 is seen at θ = 0. Sequence analysis of PROM1 located in the linkage interval identified a c.1726C>T homozygous transition in exon 15: resulting in p.Gln576X in the translated protein. This mutation is found in a homozygous state in all six affected individuals and was heterozygous in five of the six unaffected family members examined. The mutation was not detected in 192 chromosomes of unrelated control individuals of the same ethnicity and from the same region. This delineates the phenotypic characteristics of retinopathy caused by mutations in PROM1. Qingjiong Zhang, Fareeha Zulfiqar, Xueshan Xiao, Sheikh Riazuddin and J. Fielding Hejtmancik contributed equally.     

Genomewide significant linkage to stuttering on chromosome 12

Stuttering is a common and sometimes severe communication disorder, of unknown primary etiology, that exists in populations worldwide. Many types of evidence suggest a genetic contribution to stuttering; however, the complex inheritance of this disorder has hindered identification of these factors. We have employed highly inbred families to increase the power of linkage analysis of this disorder. Forty-four Pakistani families with documented or probable consanguinity, from the city of Lahore and surrounding areas, were included. Each family contained multiple cases of stuttering, which were diagnosed using the Stuttering Severity Instrument. Using the Marshfield Weber 9 marker panel, we performed a genomewide linkage scan focused on affected individuals and their parents. The analysis included 199 genotyped individuals, 144 affected and 55 unaffected. The Pedigree Relationship Statistical Test (PREST) was used to identify pedigrees that required additional specification of inbreeding. Initial nonparametric analysis gave evidence of linkage on chromosomes 1, 5, 7, and 12. Additional genotyping was performed on chromosome 12 to a 5-cM level of resolution, and 16 additional individuals were then included, bringing the number of families to 46. Analysis of the enlarged data set provided consistent evidence of linkage on chromosome 12: the S(homoz) scoring function gave a nonparametric LOD score of 4.61, and a LOD score of 3.51 was obtained using the S(all) scoring function. These results suggest that a locus on chromosome 12q may contain a gene with a large effect in this sample.     

Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.     

Tricellulin is a tight-junction protein necessary for hearing

The inner ear has fluid-filled compartments of different ionic compositions, including the endolymphatic and perilymphatic spaces of the organ of Corti; the separation from one another by epithelial barriers is required for normal hearing. TRIC encodes tricellulin, a recently discovered tight-junction (TJ) protein that contributes to the structure and function of tricellular contacts of neighboring cells in many epithelial tissues. We show that, in humans, four different recessive mutations of TRIC cause nonsyndromic deafness (DFNB49), a surprisingly limited phenotype, given the widespread tissue distribution of tricellulin in epithelial cells. In the inner ear, tricellulin is concentrated at the tricellular TJs in cochlear and vestibular epithelia, including the structurally complex and extensive junctions between supporting and hair cells. We also demonstrate that there are multiple alternatively spliced isoforms of TRIC in various tissues and that mutations of TRIC associated with hearing loss remove all or most of a conserved region in the cytosolic domain that binds to the cytosolic scaffolding protein ZO-1. A wild-type isoform of tricellulin, which lacks this conserved region, is unaffected by the mutant alleles and is hypothesized to be sufficient for structural and functional integrity of epithelial barriers outside the inner ear.     

Mesenchymal stem cells conditioned with glucose depletion augments their ability to repair-infarcted myocardium

Mesenchymal stem cells (MSCs) are an attractive candidate for autologous cell therapy, but their ability to repair damaged myocardium is severely compromised with advanced age. Development of viable autologous cell therapy for treatment of heart failure in the elderly requires the need to address MSC ageing. In this study, MSCs from young (2 months) and aged (24 months) C57BL/6 mice were characterized for gene expression of IGF‐1, FGF‐2, VEGF, SIRT‐1, AKT, p16^INK4a, p21 and p53 along with measurements of population doubling (PD), superoxide dismutase (SOD) activity and apoptosis. Aged MSCs displayed senescent features compared with cells isolated from young animals and therefore were pre‐conditioned with glucose depletion to enhance age affected function. Pre‐conditioning of aged MSCs led to an increase in expression of IGF‐1, AKT and SIRT‐1 concomitant with enhanced viability, proliferation and delayed senescence. To determine the myocardial repair capability of pre‐conditioned aged MSCs, myocardial infarction (MI) was induced in 24 months old C57BL/6 wild type mice and GFP expressing untreated and pre‐conditioned aged MSCs were transplanted. Hearts transplanted with pre‐conditioned aged MSCs showed increased expression of paracrine factors, such as IGF‐1, FGF‐2, VEGF and SDF‐1α. This was associated with significantly improved cardiac performance as measured by dp/dt_max, dp/dt_min, LVEDP and LVDP, declined left ventricle (LV) fibrosis and apoptosis as measured by Masson's Trichrome and TUNEL assays, respectively, after 30 days of transplantation. In conclusion, pre‐conditioning of aged MSCs with glucose depletion can enhance proliferation, delay senescence and restore the ability of aged cells to repair senescent infarcted myocardium.