Expression of lumican and fibromodulin following interleukin-1 beta stimulation of disc cells of the human temporomandibular joint.

Small leucine-rich repeat proteoglycans (SLRP) are present in the extracellular matrix of the temporomandibular joint (TMJ) disc. Lumican and fibromodulin, classified as class 2 SLRPs, play important roles in TMJ assembly, proliferation and inflammation. Degenerative change in the TMJ disc gives rise to the process of internal derangement (ID). In this study, we immunohistochemically examined the expression of lumican and fibromodulin in nine human TMJ specimens and examined the gene expression of both proteoglycans in cultured human TMJ disc cells under interleukin-1 beta (IL-1 ß)-stimulated conditions. An articular disc cell line was established by collagenase treatment of a TMJ disc. The subcultured cells were then incubated for 1, 3, 6, 12, 24 or 48 h under both normal and IL-1 ß (1 ng/mL) conditions. The gene expression of lumican and fibromodulin was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in the deformed disc and that IL-1 ß induces a significant increase in lumican mRNA, but not in fibromodulin mRNA, after 24∼48 h culture compared to cells cultured in the absence of IL-1 ß (P<0.05). These results indicate that lumican and fibromodulin display different behaviors and that lumican may promote regeneration of the TMJ after degeneration and deformation induced by IL-1 ß.

Expression of lumican in the articular disc of the human temporomandibular joint.

Lumican belongs to the small leucine-rich repeat proteoglycan (SLRP) gene family and has been reported to exist in the cornea, intervertebral disc and tendon. Lumican plays a significant role in the assembly and regulation of collagen fibres. The human temporomandibular joint (TMJ) disc is made up of fibrocartilage with an extracellular matrix (ECM) composed of collagen and proteoglycans. The existence and behaviour of lumican has not been studied in the human TMJ disc. Therefore, we used immunohistochemical methods to detect lumican, CD34 and vascular endothelial growth factor (VEGF) and histochemical staining with toluidine blue in 13 human TMJ specimens (10 surgically removed and 3 obtained from autopsy). In both the normal and the deformed discs we observed staining with toluidine blue. We found that the area of metachromasia inside the deformed disc was uneven and expression of lumican was strong in the areas negative for metachromasia. Staining of VEGF and CD34 inside the deformed disc was seen. We confirmed the expression of lumican in the human TMJ disc and showed that a large number of fibroblast-like cells existed in the area of strong lumican expression. These new findings about the behaviour of lumican suggest that it may play a key role in the generation of a new collagen network by fibroblast-like cells.

Mutations in the human homologue of the Drosophila segment polarity gene patched in oral squamous cell carcinoma cell lines.

In the present study, we have analyzed tumor deoxyribonucleic acid from oral squamous cell carcinoma (OSCC) cells for patched mutations using an exon-by-exon single strand conformation polymorphism assay and direct sequencing. We found two missense mutations which affected the conserved residue in the transmembrane domains of the gene product and in the intracellular loop at the C-terminal residue implicated in regulating the smoothened molecule. In addition, we demonstrated that the N-terminal fragment of sonic hedgehog (Shh-N) stimulates the growth of normal epithelial cells, the OSCC cell line, NA, and the salivary gland adenocarcinoma cell lines, HSG and HSY, which have no detectable mutation in patched. On the other hand, Shh has no effect on human SCC cells (UE, KA, KO, NI, A431 cells) that have mutations in patched. These results strongly suggest that an Shh-patched signaling is involved in the cell growth of oral epithelial cells and in the tumorigenesis of OSCCs.

High accumulation of plasminogen and tissue plasminogen activator at the flow surface of mural fibrin in the human arterial system.

PURPOSE: We assessed the fibrinolytic activity of the organized mural thrombus lining of aneurysms and prosthetic grafts. METHODS: Between May 1995 and April 1998, the full-thickness mural thrombi of aneurysms and the pseudointima lining of vascular grafts were obtained from 12 patients, ranging from 55 to 78 years in age, who underwent elective surgery. These included five aortic arch aneurysms, four abdominal aortic aneurysms, and three patent synthetic vascular grafts. The specimens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot and immunohistochemistry for human plasmin/plasminogen, tissue plasminogen activator (tPA), and fibrin degradation product (D-dimer). RESULTS: In the SDS-PAGE/immunoblot, 25- and 27-kd bands appeared specifically in experimental fibrin plates after limited digestion by plasmin and were also recognized in the mural thrombi. The presence of bands at 25 and 27 kd, which were most prominent in sections near the flow surface layer, was consistent with the hypothesis that the mural fibrin was digested by the endogenous plasmin. Apparent immunoreactivity was found at the flow surface of the masses at a thickness of 10 to 400 micrometer suggesting the presence of a plasminogen and tPA-rich layer, with D-dimer as a consequential product of fibrinolysis. CONCLUSION: The hypothesis that fibrin surfaces in the arterial system acquire fibrinolytic activity because of digestion by circulating endogenous plasmin was confirmed; this may contribute to the antithrombogenicity of these flow surfaces.

Molecular cloning and sequencing of the edeine B1 amidinohydrolase gene of Bacillus brevis TT02-8 and its expression in Escherichia coli.

The gene encoding edeine B1 amidinohydrolase from Bacillus brevis TT02-8 was cloned into Escherichia coli and its nucleotide sequence was determined. An open reading frame was identified and was found to encode a polypeptide of 289 amino acid residues with a predicted molecular weight of 32,455, which was consistent with that previously calculated for edeine B1 amidinohydrolase purified from this bacterium. Comparison of the deduced amino acid sequence of this enzyme with other amidinohydrolases revealed the highest homology to B. subtilis agmatine ureohydolase. The enzymatic activity of the protein produced in Escherichia coli was analyzed. Three histidine residues, H-112, H-137 and H-151 in the edeine B1 amidinohydrolase, which are highly conserved in amidinohydrolases, were changed to alanine by site-directed mutagenesis. Analysis of each of these mutants revealed that three histidine residues are important but not essential for the enzyme activity.

An HIV autopsy--characterization of zidovudine-resistant subtype E HIV-1 from autopsy tissue suggests the route of infection and an alternative protocol of therapy.

This CPC concerns a 47-year-old male patient with acquired immunodeficiency syndrome (AIDS). The patient became symptomatic when he developed Pneumocystis carinii pneumonia, but recovered sufficiently to be treated as an outpatient. Two years after falling ill, he developed septic shock and died within a short time. During this period, he failed to respond to HIV drugs, and there was no improvement in his immunodeficient status. The HIV retrieved from the patient's organs at autopsy was found to be type E and to have acquired resistance to Zidovudine. It was also possible to determine the route of infection. HIV treatment guidelines are continuously being revised on the basis of HIV research and the development of new treatment plans, and at the present time, when no definitive method of treatment has yet been established, it is essential for the clinician to keep abreast of the latest information. Since HIV patients are compromised hosts, it is important to diagnose and treat other infectious complications, not only complications unique to AIDS, and we have briefly described the latest HIV therapy.

Immobilization of N-carbamyl-D-amino acid amidohydrolase.

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.

A plaque hybridization assay for quantifying and cloning infectious human immunodeficiency virus type 1 virions.

A biological method was developed for quantifying and cloning of infectious virions of human immunodeficiency virus type 1 (HIV-1). Virus preparations were mixed with permissive cells for binding, and the cells were cast in an agarose gel. After incubation for 9 days viral particles released from infected cells propagating from each initially infected cell were transferred on nylon membrane and subjected to hybridization using a radioactive HIV-1 DNA probe. Infectious centers of HIV-1 were detected as hybridization spots on autoradiographs regardless of cytopathic effects or syncytium formation. Three different CD4+ cell lines (MT-4, MOLT-4 and U937) and peripheral blood mononuclear cells from healthy donors were used as recipient cells. Infectious virions were recovered from a portion of agarose gel corresponding to each hybridization spot. This assay is suitable for quantifying infectious HIV-1 virions with different cell tropisms and for investigating the relationship between the phenotype and genotype of HIV-1 at a clonal level.

[Difference of new mutation rates in dystrophin gene between deletion and duplication mutation in Duchenne and Becker muscular dystrophy].

To clarify new mutational rates in the dystrophin gene between deletion and duplication mutations, carrier diagnosis was performed on 123 mothers of probands suffered from Duchenne (DMD) and Becker (BMD) muscular dystrophy. Quantitative Southern blot analysis with cDNA probes was applied in this study. Out of 108 mothers of DMD/BMD patients with deletion mutation in dystrophin gene, 69 were carriers and 39 were non-carriers. On the other hands, all of 15 mothers of probands with duplication mutation were carriers. The fact that no new mutation occurred in oogenesis in the families with duplication mutations in dystrophin gene indicates that duplications arise in spermatogenesis. The risk of the mother of an isolated case of DMD/BMD with duplication mutation of being a carrier is significantly higher than the estimated risk based on the equality of new mutation in oogenesis and spermatogenesis.

Identification of carriers of Duchenne/Becker muscular dystrophy by a novel method based on detection of junction fragments in the dystrophin gene.

We developed a Southern blotting based method that uses rare cutting restriction endonucleases and electrophoresis of single stranded DNA to detect junction fragments resulting from the rearranged dystrophin gene. By conventional Southern blot hybridisation, no junction fragments were detected in 27 unrelated patients with Duchenne (DMD) or Becker (BMD) muscular dystrophy, who had 20 deletions and seven duplications in the dystrophin gene. With our new method, junction fragments were detected in 21 of these 27 patients. When the junction fragments were used as markers, five carriers were unequivocally diagnosed among six females from two families of DMD/ BMD patients. This novel method allows simple and definitive identification of carriers with risk factors for DMD/BMD without using quantitative Southern blot hybridisation.

Quantitative Southern blot analysis in the dystrophin gene of Japanese patients with Duchenne or Becker muscular dystrophy: a high frequency of duplications.

Eighty-four unrelated patients with Duchenne or Becker muscular dystrophy in Japan were studied by quantitative Southern blot analysis with dystrophin cDNA probes. We found partial deletions and duplications in 47 (56%) and 12 (14%) cases respectively by HindIII digestion. The duplications were confirmed by BglII digestion and densitometric scanning. The frequency of duplications in this study is significantly higher than those previously reported. This may be because of the small sample number, the racial difference, or our quantitative methods. Our results suggest that attempts to detect duplications are important for a precise diagnosis. Both deletions and duplications clustered at the two hot spots as reported previously. Six cases were exceptions to the 'reading frame hypothesis'. We detected three types of HindIII RFLP. Based on the results of one duplication case, we propose a revised sequential order of exons in the cDNA10 region of the dystrophin gene.