Identification of COL4A5 defects in Alport's syndrome by immunohistochemistry of skin.

BACKGROUND: The COL4A3-COL4A4-COL4A5 network in the glomerular basement membrane is affected in the inherited renal disorder Alport's syndrome (AS). Approximately 85% of the AS patients are expected to carry a mutation in the X-chromosomal COL4A5 gene and 15% in the autosomal COL4A3 and COL4A4 genes. The COL4A5 chain is also present in the epidermal basement membrane (EBM). It is predicted that approximately 70% of the COL4A5 mutations prevent incorporation of this chain in basement membranes. METHODS: We investigated whether or not COL4A5 defects could be detected by immunohistochemical analysis of the EBM. Punch skin biopsies were obtained from 22 patients out of 17 families and two biopsy specimens from healthy males were used as controls. RESULTS: In four cases with the COL4A5 frameshift or missense mutations, the COL4A5 chain was either lacking from the EBM (male) or showed a focally negative pattern (female). In three other patients with a COL4A5 missense mutation, a COL4A3 and a COL4A4 mutation, respectively, the COL4A5 staining was normal. A (focally) negative EBM-COL4A5 staining was found in three patients of six families with a diagnosis of AS and in one family of a group of four families with possible AS. CONCLUSIONS: The (focal) absence of COL4A5 in the EBM of skin biopsy specimens can be used for fast identification of COL4A5 defects. Combined with polymorphic COL4A5 markers, both postnatal and prenatal DNA diagnosis are possible in the family of the patient.

Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant.

Recently we described a protein, smoothelin, that has been exclusively found in smooth muscle cells (SMC). The human cDNA has been cloned from a colon cDNA library and the putative protein sequence was deduced. Smoothelin does not belong to a known protein family but shows a partial homology with members of the spectrin family. Transfection studies revealed that smoothelin has an affinity for actin and is either capable of forming filamentous structures or colocalizes with such structures. The protein is expressed in visceral as well as vascular tissues of all vertebrate classes. A study on the distribution of smoothelin in the vascular and placental system showed that smoothelin expression was largely restricted to the muscular pulsating blood vessels. Therefore, we hypothesized that smoothelin is expressed in contractile SMC only (36, 37). No expression of smoothelin was observed in established cell lines of SMC. In tissue explants smoothelin mRNA concentration decreases to undetectable levels within 12 hours after dissection as was in general the case in primary cell cultures. Here we report on continued smoothelin expression for several passages observed in a human prostate primary cell culture system. Smoothelin was demonstrated to colocalize with actin stress fibers but not with desmin filaments. This culture system offers opportunities to study the cytological localization of smoothelin, interactions with other proteins and should provide a system to test the promoter of the smoothelin gene. On immunoblots the molecular weight of smoothelin differed between visceral and vascular smooth muscle tissue with apparent molecular weights of respectively 59 kDa and 94 kDa. There is no evidence for the existence of another gene coding for the 94 kDa smoothelin. Thus, posttranslational modification, alternative splicing and dual promoter control are the alternatives for the expression of two isoforms of smoothelin.

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of approximately 40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, beta-spectrin and alpha-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (approximately 1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.

Titin expression as an early indication of heart and skeletal muscle differentiation in vitro. Developmental re-organisation in relation to cytoskeletal constituents.

Established myogenic cell lines of different species and tissue origin have been used to study expression and organisation of muscle-specific proteins during differentiation. Furthermore, primary cultures of rat myocard cells were used to examine these same processes during dedifferentiation. In particular, we were interested in the general mechanism that underlies the changes in the supramolecular organisation of titin during in vitro myogenesis. It became obvious that in the differentiating muscle cell cultures the redistribution of desmin, actin and myosin in a typical, differentiation state dependent fashion, always showed a certain delay when compared to titin. The sequence of changes in the assembly of cytoskeletal and sarcomeric structures observed during differentiation of the cell lines was reversed during the process of dedifferentiation in cultured rat myocard cells. These results all indicate that titin is an early marker of myogenic differentiation, both in vivo and in vitro, and the typical reorganisation of this giant molecule is independent of species or muscle cell type.

Cultured pig rhabdomyosarcoma cells with a deletion of the Xq24-qter chromosome region: an immunochemical and cytogenetic characterization.

A pig rhabdomyosarcoma cell line (PRUM59) was established, and the immuno(histo)chemical and cytogenetic characterization of these cells was determined. At various swine farms in the Netherlands, pigs were observed that had solitary or multiple skin nodules, which were diagnosed as rhabdomyosarcomas. Cells of a tumor derived from a 3.5-week-old female pig were cultured for immunochemical and cytogenetic analyses. The cell line had characteristic features of undifferentiated muscle cells, similar to those observed in tumor tissue sections; they contained titin, a high-molecular weight protein specific for striated muscle, as dot-like aggregates and as filaments, desmin filaments and cross-striations, smooth muscle actin stress fibers, and vimentin filaments. The cells stained positively for striated muscle actin and tropomyosin as well. The immunohistochemical staining results were supported by results of immunoblotting experiments. Karyotyping of the cells revealed a deletion of a major part of Xq24-qter, a part of the long arm of 1 of the 2 X chromosomes. The other X chromosome and all autosomes appeared to be normal.

Ambient and biological monitoring of cokeoven workers: determinants of the internal dose of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAH) were measured in the breathing zone air of 56 battery workers at two cokeovens during three consecutive days. The concentration of total PAH ranged up to 186 micrograms/m3. Preshift and end of shift urine samples were collected to determine 1-hydroxypyrene, a metabolite of pyrene. Control urine samples were available from 44 workers in the shipping yard of a hot rolling mill. The median values of 1-hydroxypyrene in urine of smoking and non-smoking controls were 0.51 and 0.17 mumol/mol creatinine, respectively. Concentrations of 1-hydroxypyrene up to 11.2 mumol/mol were found in the urine of the cokeoven workers. At the start of the three day working period after 32 hours off work, the 1-hydroxypyrene concentrations were four times higher and at the end of the working period 10 times higher compared with control concentrations. Excretion of 1-hydroxypyrene occurred with a half life of 6-35 hours. Both the ambient air monitoring data and the biological monitoring data showed that the topside workers were the heaviest exposed workers. The relation between air monitoring data and biological monitoring data was not strong. Multiple regression analysis was performed to identify determinants of the internal dose. The combination of exposure and smoking amplify each other and the use of a protective airstream helmet decreases the internal dose. An effect of alcohol consumption and the use of medication on the toxicokinetics of pyrene was not found.