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Biomedical subjects

L Sakai

Publications and source records attributed to L Sakai.

15 recordsLinked to original sources
PubMed2001

Regulation of limb patterning by extracellular microfibrils.

To elucidate the contribution of the extracellular microfibril-elastic fiber network to vertebrate organogenesis, we generated fibrillin 2 (Fbn2)-null mice by gene targeting and identified a limb-patterning defect in the form of bilateral syndactyly. Digit fusion involves both soft and hard tissues, and is associated with reduced apoptosis at affected sites. Two lines of evidence suggest that syndactily is primarily due to defective mesenchyme differentiation, rather than reduced apoptosis of interdigital tissue. First, fusion occurs before appearance of interdigital cell death; second, interdigital tissues having incomplete separation fail to respond to apoptotic clues from implanted BMP-4 beads. Syndactyly is associated with a disorganized matrix, but with normal BMP gene expression. On the other hand, mice double heterozygous for null Fbn2 and Bmp7 alleles display the combined digit phenotype of both nullizygotes. Together, these results imply functional interaction between Fbn2-rich microfibrils and BMP-7 signaling. As such, they uncover an unexpected relationship between the insoluble matrix and soluble factors during limb patterning. We also demonstrate that the Fbn2- null mutation is allelic to the recessive shaker-with-syndactyly (sy) locus on chromosome 18.

Alleles↗
PubMed2001

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.

Aggrecans↗
PubMed2001

Vasculitis following treatment of rheumatoid arthritis with extracorporeal staphylococcal protein a immunoadsorption column (prosorba).

We report a case of vasculitis after Prosorba treatment in a patient with rheumatoid arthritis. The patient is a 66-year-old white male with long standing rheumatoid arthritis and hepatitis B. He was treated with the standard regimen for Prosorba treatment. He improved and met criteria for an American College of Rheumatology (ACR) 20% response. While on therapy he developed a nonhealing ulcer. Approximately 2 weeks after treatment was completed, he developed palpable purpura and mononeuritis multiplex. Deep dermal biopsy confirmed the presence of both small and medium vessel vasculitis. Nerve conductions studies were consistent with neuropathic conduction delays. He was treated with 1mg/kg/day of oral prednisone. Prosorba has been reported to cause leukocytoclastic vasculitis during treatment, but has not been noted to involve medium sized vessels. This patient's history and presentation are most consistent with rheumatoid arthritis associated vasculitis, though the Prosorba treatment cannot be ruled out as a cause or a contributing factor. Importantly, although Prosorba treated his synovitis, it did not prevent concomitant vasculitis.

Journal Article↗
PubMed1998

Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.

Adult↗
PubMed1998

Evidence for furin-type activity-mediated C-terminal processing of profibrillin-1 and interference in the processing by certain mutations.

Fibrillin-1 is a major component of the 10 nm microfibrils of the extracellular matrix (ECM). It is synthesized as an approximately 350 kDa precursor molecule, profibrillin-1, which is proteolytically processed into its biologically active approximately 320 kDa form. Furin, a calcium-dependent endoprotease of the subtilisin family, which is known to be the processing enzyme for a variety of proproteins, is believed to be responsible for the N-terminal proteolytic cleavage of profibrillin-1. In this article we provide several lines of evidence that the C-terminal trimming of profibrillin-1 also occurs via a furin-type activity. Edman degradation of a small recombinant C-terminal subdomain of fibrillin-1 revealed complete processing of the peptide immediately after the tribasic recognition sequence (R-X-K/R-R) for furin. In vitro expression experiments using another recombinant construct consisting of the C-terminal half of fibrillin-1 indicated that disruption of the putative recognition sequence for furin by site-directed mutagenesis drastically impairs proteolytic processing of the propeptide. In addition, our results suggest that the N-terminal half of fibrillin-1 is necessary for its incorporation into the ECM.

Amino Acid Sequence↗
PubMed1994

Abnormalities of fibrillin in acquired cutis laxa.

BACKGROUND: Degeneration of elastic tissue in acquired cutis laxa has been previously described, but microfibrils have not been adequately studied. OBJECTIVE: We determined whether the microfibrillar component of elastic tissue is affected in skin of a patient with acquired cutis laxa. METHODS: Lesional skin was examined with indirect immunofluorescence and immunoelectron microscopy with antibodies to fibrillin. RESULTS: Indirect immunofluorescence showed a reduction in the distribution of fibrillin in the papillary dermis, where there was loss of the usual pattern of microfibrils perpendicular to the epidermis. Immunoelectron microscopy showed a typical distribution of elastic microfibrils around elastin of normal skin. In skin affected by cutis laxa microfibrils appeared morphologically normal but appeared less frequently in selected sites. CONCLUSION: The microfibrillar component of elastic fibers was reduced in the papillary dermis of this patient with acquired cutis laxa.

Actin Cytoskeleton↗
PubMed1994

Mutations in the fibrillin gene responsible for dominant ectopia lentis and neonatal Marfan syndrome.

Mutations in the gene coding for fibrillin on chromosome 15 (FBN1) are known to cause Marfan syndrome (MFS). A related disorder, dominant ectopia lentis (EL), has also been linked genetically to this locus. We now describe ten novel mutations of FBN1 resulting in strikingly different phenotypes. In addition to classic MFS, FBN1 mutations also give rise to EL and a severe neonatal form of MFS. Interestingly, the neonatal MFS mutations are clustered in one particular region of FBN1, possibly providing new insights into genotype-phenotype comparisons.

Adolescent↗
PubMed1993

Autosomal dominant Marfan-like connective-tissue disorder with aortic dilation and skeletal anomalies not linked to the fibrillin genes.

We describe a large family with a connective-tissue disorder that exhibits some of the skeletal and cardiovascular features seen in Marfan syndrome. However, none of the 19 affected individuals displayed ocular abnormalities and therefore did not comply with recognized criteria for this disease. These patients could alternatively be diagnosed as MASS (mitral valve, aorta, skeleton, and skin) phenotype patients or represent a distinct clinical entity, i.e., a new autosomal dominant connective-tissue disorder. The fibrillin genes located on chromosomes 15 and 5 are clearly involved in the classic form of Marfan syndrome and a clinically related disorder (congenital contractural arachnodactyly), respectively. To test whether one of these genes was also implicated in this French family, we performed genetic analyses. Blood samples were obtained for 56 family members, and four polymorphic fibrillin gene markers, located on chromosomes 15 (Fib15) and 5 (Fib5), respectively, were tested. Linkage between the disease allele and the markers of these two genes was excluded with lod scores of -11.39 (for Fib15) and -13.34 (for Fib5), at theta = .001, indicating that the mutation is at a different locus. This phenotype thus represents a new connective-tissue disorder, overlapping but different from classic Marfan syndrome.

Adult↗
PubMed1991

Middermal elastolysis. Report of a case and immunohistochemical studies on the dermal distribution of fibrillin, vitronectin and amyloid P component.

A 39-year-old woman with demarcated wrinkled areas, histologically characterized by absence of elastic fibers in the middle and upper reticular dermis, is described. Immunoreactivity of vitronectin and amyloid P component, present at the periphery of elastic fibers in normal skin in adults, was absent from the middermis of lesional skin as were orcein stained fibers. C9 neoantigen immunoreactivity, associated with elastic fibers in sun-exposed skin of middle-aged and elderly individuals, was present in conjunction with elastic fibers in papillary and lower reticular dermis in lesional skin but was absent in the middermis. In contrast, a fibrillin immunoreactive network was present throughout the dermis, indicating that the elastin-associated microfibrils are retained in the absence of amorphous elastin in lesional skin of middermal elastolysis.

Adult↗
PubMed1990

IgA immunoreactive deposits collocal with fibrillin immunoreactive fibers in dermatitis herpetiformis skin.

The IgA immunoreactive granules or fibrils, characteristically found in dermal papillae of patients with dermatitis herpetiformis, were previously reported to be associated with microfibrillar bundles. Recently, fibrillin, a component of such 8-12 nm microfibrils, was identified. In normal skin, the fibrillin immunoreactive microfibrils are present at the periphery of elastic fibers and are also present without concomitant amorphous elastin in the dermal papillae close to the lamina densa. The localization of the IgA immunoreactive material in the dermal papillae of 17 patients with dermatitis herpetiformis was compared with the distribution of the fibrillin immunoreactive fiber network. Immunofluorescence methods using FITC- and TRITC-labelled antibodies, an avidin-biotin-peroxidase complex technique, and standard elastin staining procedures, were used in several sequential and double staining procedures. In 13 specimens, in which the IgA reactivity was granular, most of the granules were located at the sites of fibrillin-reactive structures. As it could not be excluded that the collocality was coincidental, it could not be ascertained whether the IgA granules were in fact related to the fibrillin immunoreactive fibers in these specimens. However, in 4 specimens with both granular and fibrillar IgA immunoreactive deposits, these were clearly related to and located at the sites of fibrillin-reactive fibrils in the dermal papillae. The results confirm earlier reports of an association of IgA reactive deposits with microfibrillar bundles in dermatitis herpetiformis skin, though the possibility of their binding to other extracellular matrix component(s) has not been ruled out. The findings suggest that fibrillin may be the structural component (or one of them) to which IgA reactive deposits bind in the skin of patients with dermatitis herpetiformis.

Complement C3↗
PubMed1990

Immunohistochemical studies on fibrillin in amyloidosis, lichen ruber planus and porphyria.

The pathogenesis of macular amyloidosis and lichen amyloidosis remains unsolved and the primary amyloid fibril protein(s) has not yet been identified. Ultrastructural association of skin amyloid with elastin associated microfibrils has been noted earlier. The presence of fibrillin in conjunction with such microfibrils was recently demonstrated immunohistochemically. The presence of fibrillin immunoreactivity in the amyloid deposits in skin biopsies from 3 patients with macular amyloidosis and 3 patients with lichen amyloidosis was studied, using monoclonal anti-fibrillin antibodies. For comparison, skin specimens were studied from five patients with lichen ruber planus, four patients with erythropoietic protoporphyria and from a patient with myeloma-associated cutaneous amyloidosis. Renal specimens from two cases of the amyloid A type of renal amyloidosis also were investigated. There was no immunostaining either of the keratin bodies in specimens of lichen ruber planus, the cutaneous PAS-positive vascular deposits in patients with erythropoietic protoporphyria, or the amyloid deposits in specimens of systemic amyloidosis and it was faint or absent in amyloid deposits in the specimens from patients with lichen amyloidosis. In contrast, distinct fibrillin immunoreactivity could be demonstrated in amyloid deposits in specimens from patients with macular amyloidosis. It was sometimes absent in deposits located in the upper part of the papillary dermis, close to the dermal epidermal junction zone, while consistently strong in deposits located lower down in the dermis. The results suggest that fibrillin or part of the fibrillin molecule may be present in some of the amyloid deposits in specimens of macular amyloidosis.

Amyloidosis↗
PubMed1987

Pulmonary fibroblasts: a model system for studying elastin synthesis.

Pulmonary interstitial fibroblasts were isolated from 4-day neonatal rat lung and cultured for varying periods of time after first passage. The ability of the cells to synthesize both soluble and insoluble elastin was examined by biochemical, immunological, and ultrastructural techniques. The cells synthesize a tropoelastin of 75,000 daltons which is cross-linked in the extracellular matrix to form sufficient amounts of insoluble elastin to be characterized directly by amino acid analysis. Although the cells produce a soluble form of elastin immediately after confluency and the deposition in the matrix begins shortly thereafter, the actual chemical definition of alkali-resistant elastin does not occur until 4 weeks after confluency. The apparent molecular weight of tropoelastin synthesized by the cells agrees with the molecular size of tropoelastin found in organ culture of neonatal lungs. The pulmonary fibroblast cultures provide an important model system for examining the regulation and response of elastin synthesis in lung interstitium.

Animals↗
PubMed1981

Acute perforation of sigmoid diverticula.

Forty patients underwent operative therapy for acute perforation of colonic diverticula. The group I patients had some localization of infection, group II had generalized feculent-purulent contamination with free perforation, and group III had free perforation and were treated with chronic steroids for various medical disorders. The retrospective evaluation was directed at evaluating the effect on survival of methods to treat the hole in the colon and methods to treat peritoneal contamination. It is concluded that control of the perforation in the bowel and aggressive peritoneal irrigation may decrease the high morbidity and mortality rates.

Adult↗
PubMed1980

Spontaneous and shock-associated ischemic colitis.

Ischemic changes in the colon that progress to gangrene present diagnostic and therapeutic difficulties associated with poor survival. During the past 10 years, 36 patients with colonic ischemia were treated. Two clinical groups were evident. The spontaneous ischemic colitis group (type I) included 17 patients who were well before the onset of gastrointestinal symptoms. The cause of type I ischemic colitis is not apparent; it is attributed to occlusive or nonocclusive ischemia. The other group (type II) included 19 patients who developed ischemia of the colon associated with shock secondary to various disease processes. Radiographic evidence of distended bowel correlated well with full-thickness necrosis as determined clinically or pathologically. Twenty-one patients died, for a mortality rate of 58 percent. While full-thickness gangrene was fatal in 71 percent of the patients, mucosal necrosis only was associated with an 88 percent survival rate. The results of treatment should improve with an increased awareness of ischemic colitis, earlier appropriate operative intervention, and more appropriate use of ileostomy and colostomy.

Aged↗