Usher syndrome type IIa (USHIIa) is an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa. This disorder maps to human chromosome 1q41. Recently, mutations in USHIIa patients were identified in a novel gene isolated from this chromosomal region. The USH2A gene encodes a protein with a predicted molecular weight of 171.5 kD and possesses laminin epidermal growth factor as well as fibronectin type III domains. These domains are observed in other protein components of the basal lamina and extracellular matrixes; they may also be observed in cell-adhesion molecules. The intron/exon organization of the gene whose protein we name "Usherin" was determined by direct sequencing of PCR products and cloned genomic DNA with cDNA-specific primers. The gene is encoded by 21 exons and spans a minimum of 105 kb. A mutation search of 57 independent USHIIa probands was performed with a combination of direct sequencing and heteroduplex analysis of PCR-amplified exons. Fifteen new mutations were found. Of 114 independent USH2A alleles, 58 harbored probable pathologic mutations. Ten cases of USHIIa were true homozygotes and 10 were compound heterozygotes; 18 heterozygotes with only one identifiable mutation were observed. Sixty-five percent (38/58) of cases had at least one mutation, and 51% (58/114) of the total number of possible mutations were identified. The allele 2299delG (previously reported as 2314delG) was the most frequent mutant allele observed (16%; 31/192). Three new missense mutations (C319Y, N346H, and C419F) were discovered; all were restricted to the previously unreported laminin domain VI region of Usherin. The possible significance of this domain, known to be necessary for laminin network assembly, is discussed in the context of domain VI mutations from other proteins.

We have shown previously that alpha-dystroglycan with a molecular mass of 120 kDa is a Schwann cell receptor of laminin-2, the endoneurial isoform of laminin comprised of the alpha2, beta1, and gamma1 chains. In this paper, we show that Schwann cell alpha-dystroglycan is also a receptor of agrin, an acetylcholine receptor-aggregating molecule having partial homology to laminin alpha chains in the C terminus. Immunochemical analysis demonstrates that the peripheral nerve isoform of agrin is a 400-kDa component of the endoneurial basal lamina and is co-localized with alpha-dystroglycan surrounding the outermost layer of myelin sheath of peripheral nerve fibers. Blot overlay analysis demonstrates that both endogenous peripheral nerve agrin and laminin-2 bind to Schwann cell alpha-dystroglycan. Recombinant C-terminal fragment of the peripheral nerve isoform of agrin also binds to Schwann cell alpha-dystroglycan, confirming that the binding site for Schwann cell alpha-dystroglycan resides in the C terminus of agrin molecule. Furthermore, the binding of recombinant agrin C-terminal fragment to Schwann cell alpha-dystroglycan competes with that of laminin-2. All together, these results indicate that alpha-dystroglycan is a dual receptor for agrin and laminin-2 in the Schwann cell membrane.

An improved myringoplasty technique utilizing fibrin glue and carried out through the external auditory canal was recently introduced. This endoscopic-assisted technique allows exquisite views and avoids blind surgical procedures, thereby expanding the indications for minimally invasive myringoplasty. This technique was applied to patients in whom, due to the curved external auditory canal, the margin of the perforation of the tympanic membrane was not visible with an operating microscope. We summarized the results of 22 endoscopic-assisted myringoplasties and concluded that this technique provides satisfactory results both in the success rate of perforation closure and in hearing outcome.

Dystroglycan is encoded by a single gene and cleaved into two proteins ␣-and ␤-dystroglycan by posttranslational processing. Recently, ␣-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein ␤-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of ␣-and ␤-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of ␣-dystroglycan, but not ␤-dystroglycan, in the culture medium, suggesting secretion of ␣-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against ␣-dystroglycan was shown previously to inhibit the binding of laminin-1 to ␣-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of ␣-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells.

We performed quantification of IL 2, IL 4, IL 6, IL 8, IL 10, GM-CSF, IFN gamma, and TNF alpha in human dermal wounds for wound age estimation. The proliferation of dermal cells and infiltration of inflammatory cells were also analyzed. Neutrophils and macrophages were detected from 2 h post-injury, and strong infiltrations were seen at 33-49 h. T and B lymphocytes also infiltrated simultaneously from 71 h. Strong proliferation of fibroblasts were shown from 246 h, and thickening of the epidermis from 71 h. IL 10, GM-CSF, IFNgamma, and TNF alpha increased from the early phase of dermal wound healing, IL 6 exclusively in the middle phase, IL 2, IL 4, and IL 8 from the middle phase to the late phase. Among the cytokines analyzed in the present study, IL 6, IL 8, IFNgamma, and TNF alpha were strongly expressed. Results of the present study suggest that multiplex cytokine analysis at the wound site can be useful for wound age estimation. In addition, multiplex data obtained from the same sample with a single method would demonstrate more accurate interactions of cytokines during dermal wound healing. Although the present study was oriented to practical forensic pathology, the data obtained would be informative for various fields of medicine.