Biomedical subjects

E Sakashita

Publications and source records attributed to E Sakashita.

15 recordsLinked to original sources

PubMed2001

Development of a novel polycationic adsorbent for cryogel removal.

Cryogel, prevalent in the plasma of rheumatism patients, is a plasma fibronectin (pFN)-extra domain A containing FN (EDA(+)FN)-fibrinogen (Fbg) complex formed by adding heparin (HP) at a low temperature (4 degrees C). Although EDA(+)FN does not usually exist in normal plasma, its prevalence in rheumatic patients causes cryogelation in plasma. Removal of cryogel is thus a promising and novel approach to treating rheumatism. As HP-EDA(+)FN aggregate, which is induced by the main component of cryogel, is considered to be an anion, cationic materials capable of eliminating this anionic conjugate were innovated in this study. We found that an amino group density of 100-130 micromol/g (dry weight) of adsorbents prompted selective adsorption of the EDA(+)FN-HP complex. Elimination of EDA(+)FN as high as 80% accompanied by removal of the components of total FN (pFN) (10%) and Fbg (10%) in the model patient plasma was established.

Adsorption↗

PubMed2001

Cryogelation in vitro.

Cryogel is a physical gel formed by the heterophilic aggregation of extra domain A containing fibronectin (EDA(+)FN), plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep). Cryogelation is controlled by the interactions between each aggregate and the amount of aggregates. Therefore, the present study attempted to elucidate these properties by studying turbidity (tau). Although only Fbg formed a self-aggregate under low temperatures, from the temperature dependence of tau, the amount of aggregate in three-element (pFN/Fbg/Hep) solution surpassed that of the EDA(+)FN/Fbg/Hep system. The optimal condition for cryogelation was afforded by a solution with Fbg/EDA(+)FN/pFN/Hep expressed in the molar ratio of 12:0.04:0.79:1. This cryogel structure in solution was probably formed via structural changes induced by pFN in Fbg. The structural change in Fbg was examined by circular dichroism under optimal conditions. This concept was based on observations of the direct transmission scanning electron microscopy of a cryogel. The EDA(+)FN/pFN/Fbg/Hep aggregates displayed a network structure that manifested particulate crosslinkage. Cryogelation, a phenomenon related to induction of rheumatoid arthritis in humans, was facilitated by both the EDA(+)FN-Hep interaction and the structural changes of Fbg induced by pFN.

Circular Dichroism↗

PubMed2001

Experimental study using heparin-immobilized adsorbent of EDA(+)fibronectin.

EDA(+)fibronectin, which might participate in the pathogenesis and/or progress of immune diseases, is efficiently removed from plasma by cryofiltration; however, cryofiltration removes not only EDA(+)fibronectin, but also other proteins. We thus developed a new adsorbent by using its high affinity with heparin. The purpose of this study was to evaluate the efficacy of the adsorbent of EDA(+)fibronectin (OHC-20) in experimental arthritis. The experimental arthritis was induced by injection of 0.5 mg of Mycobacterium butyricum in Lewis rats. Rats were divided into 4 groups; 1 nontreatment group, and 3 treatment groups. Adsorption therapy in treatment groups was performed three times: on Days 1, 3, and 5 in Group A; Days 7, 9, and 11 in Group B; and Days 13, 15, and 17 in Group C. The walking postures of rats improved from dragging to walking on tiptoe, and the increase of hind-foot volume was suppressed in Groups B and C. We conclude that heparin-immobilized adsorbent might be promising for immune diseases.

Adsorption↗

PubMed2000

Increased extra domain-A containing fibronectin and hepatic dysfunction during septic response: an in vivo and in vitro study.

A massive inflammatory reaction resulting from systemic cytokine release is the common pathway underlying sepsis or multiple organ dysfunction. The role of extra domain sequence A-containing fibronectin (EDA+FN) formation during the septic response is not known. The present study investigates the role of EDA+FN during the septic response under in vitro and in vivo conditions. The direct effects of interleukin-1, interleukin-6, and tumor necrosis factor-alpha on EDA+FN production were evaluated in primary cultured human hepatocytes and fibroblasts. Serial plasma EDA+FN levels were measured using an enzyme-linked immunosorbent assay in 24 patients who developed postoperative sepsis following general abdominal surgery of which there were 17 survivors and 7 non-survivors. EDA+FN secretion was significantly increased in cultured hepatocytes but not fibroblasts at 24 and 48 h following exposure to IL-1 compared to controls. In the clinical setting plasma EDA+FN levels in non-survivors were significantly higher than in survivors. Moreover, the EDA+FN levels were correlated closely with liver function tests. EDA+FN levels may represent a specific marker of vascular injury or systemic inflammatory response syndrome that is associated with an adverse clinical outcome.

APACHE↗

PubMed1999

Cytoplasmic localization is required for the mammalian ELAV-like protein HuD to induce neuronal differentiation.

BACKGROUND: ELAV-like neuronal RNA-binding proteins are highly conserved in many neurone-containing organisms and have been implicated in neuronal development and differentiation. RESULTS: Mammalian neurone-specific ELAV-like Hu proteins (HuB, HuC and HuD) and Drosophila ELAV, but not HuR, were found to induce neurite outgrowth when over-expressed in rat PC12 cells. Functional analysis of HuD deletion mutants demonstrated the importance of two conserved RNA-binding domains (RBDs 1 and 3) and the indispensability of the linker region between RBDs 2 and 3 for the neurite-inducing activity. Further analyses suggested the importance of nucleocytoplasmic shuttling of HuD mediated by a novel nuclear export signal (NES) sequence in the linker region for the neurite-inducing activity. Moreover, two HuD deletion mutants containing the linker region dominantly inhibited the wild-type neurite-inducing activity, although they had no neurite-inducing activity per se, suggesting that saturable intracellular trafficking mediated by the linker region is required for the neurite induction by HuD. Interestingly, the same dominant negative mutants significantly inhibited retinoic acid-induced neuronal differentiation of mouse embryonal carcinoma P19 cells. CONCLUSIONS: Our results suggest the presence of a novel NES in neurone-specific Hu proteins and the importance of their cytoplasmic localization, through nucleocytoplasmic shuttling, for the initiation of neuronal differentiation.

Amino Acid Sequence↗

PubMed1999

Different responses to surgical stress between extra domain A+ and plasma fibronectins.

1. Fibronectins (FN) are believed to have a role in haemorheological perturbation associated with tissue damage. Fibronectins exist in two antigenically related forms, plasma (p) and cellular fibronectin, which has the extra domain sequences A (EDA) or B (EDB). The present study was designed to determine changes in plasma p-FN and EDA + FN under different types of surgical stress. 2. Sixty-two patients were divided into three groups: (i) group A, 33 patients undergoing hepato-pancreato-biliary surgery; (ii) group B, 19 patients undergoing laparoscopic cholecystectomy; and (iii) group C, 10 patients with postoperative complications. Plasma FN and EDA + FN levels were measured in these patients undergoing different types of surgical operation and either with or without liver cirrhosis using an enzyme-linked immunosorbent assay. 3. After surgery, a significant decrease in p-FN levels and a significant increase in EDA + FN levels was observed in all patient group compared with pre-operative levels. The duration of increased EDA + FN levels, but not p-FN levels, in group A patients was significantly longer than in group B patients. Although changes in p-FN levels between patients with and without liver cirrhosis were significantly different, there were no significant differences in the EDA + FN levels between these two patient groups. 4. In conclusions, EDA + FN and p-FN levels were found to exhibit opposite responses to surgical stress. Furthermore, with greater surgical stress, greater increases in EDA + FN levels were seen. The presence of liver cirrhosis had no significant effect on EDA + FN levels during the perioperative period; however, p-FN levels were significantly affected. 5. Thus, it is suggested that plasma EDA + FN levels reflect the magnitude of surgical stress more closely than do p-FN levels.

Adult↗

PubMed1998

Development of the selective adsorbent for EDA containing fibronectin using heparin immobilized cellulose.

We have noticed that extra domain A containing fibronectin (EDA(+)FN) is closely related to inflammatory diseases and accordingly investigated a selective adsorbent to remove EDA(+)FN, which may increase the severity of the symptoms, from the plasma of rheumatoid arthritis (RA) patients. Three types of heparin immobilized cellulose (HC) were prepared and their adsorptive properties were evaluated by batchwise adsorption tests. The results showed that EDA(+)FN was adsorbed more efficiently than total FN on HC. In particular HC in which heparin was immobilized to amino residue had an excellent capacity. The adsorption rates of EDA(+)FN, total FN and antithrombin-III were 89, 15 and 17% respectively. This adsorbent is extremely useful in selectively removing EDA(+)FN from plasma.

Adsorption↗

PubMed1996

Two different RNA binding activities for the AU-rich element and the poly(A) sequence of the mouse neuronal protein mHuC.

HuC is one of the RNA binding proteins which are suggested to play important roles in neuronal differentiation and maintenance. We cloned and sequenced cDNAs encoding a mouse protein which is homologous to human HuC (hHuC). The longest cDNA encodes a 367 amino acid protein with three RNA recognition motifs (RRMs) and displays 96% identity to hHuC. Northern blot analysis showed that two different mRNAs, of 5.3 and 4.3 kb, for mouse HuC (mHuC) are expressed specifically in brain tissue. Comparison of cDNA sequences with the corresponding genomic sequence revealed that alternative 3' splice site selection generates two closely related mHuC isoforms. Iterative in vitro RNA selection and binding analyses showed that both HuC isoforms can bind with almost identical specificity to sequences similar to the AU-rich element (ARE), which is involved in the regulation of mRNA stability. Functional domain mapping using mHuC deletion mutants showed that the first RRM binds to ARE, that the second RRM has no RNA binding activity by itself, but facilitates ARE binding by the first RRM and that the third RRM has specific binding activity for the poly(A) sequence.

Alternative Splicing↗

PubMed1996

Adherence of synovial cells on EDA-containing fibronectin.

OBJECTIVE: To investigate the role of EDA-containing fibronectin (EDA+ FN), a splice variant of FN detectable in association with cellular transformation, in the adherence of synovial cells (SC) on rheumatoid cartilage surface. METHODS: The number of SC adherent on cartilage slices or on culture plates containing either EDA+ FN or plasma FN (pFN) was enumerated under a phase-contrast microscope. The portion of the FN molecule responsible for adherence of SC onto EDA+ FN was investigated by inhibition studies using antibodies or peptide fragments. RESULTS: SC adhered more strongly on the surfaces containing EDA+ FN than on those containing pFN (P < 0.01). When monoclonal antibodies against the EDA or the carboxyl-terminal heparin-binding (Hep2) domains were used, adhesion of SC onto EDA+ FN was reduced to a level comparable with that onto pFN. FN fragments containing Hep2 or heparan sulfate inhibited the adhesion of SC onto EDA+ FN. Treatment of SC with heparitinase, but not heparinase, reduced the adhesion of SC onto EDA+ FN. CONCLUSION: EDA+ FN enhances adherence of SC on the matrix via the Hep2 region of EDA+ FN.

Alternative Splicing↗

PubMed1996

Regulation of the gene Sex-lethal: a comparative analysis of Drosophila melanogaster and Drosophila subobscura.

The Drosophila gene Sex-lethal (Sxl) controls the processes of sex determination and dosage compensation. A Drosophila subobscura genomic fragment containing all the exons and the late and early promotors in the Sxl gene of D. melanogaster was isolated. Early Sxl expression in D. subobscura seems to be controlled at the transcriptional level, possibly by the X:A signal. In the region upstream of the early Sxl transcription initiation site are two conserved regions suggested to be involved in the early activation of Sxl. Late Sxl expression in D. subobscura produces four transcripts in adult females and males. In males, the transcripts have an additional exon which contains three translational stop codons so that a truncated, presumably nonfunctional Sxl protein is produced. The Sxl pre-mRNA of D. subobscura lacks the poly-U sequence presented at the polypirimidine tract of the 3' splice site of the male-specific exon present in D. melanogaster. Introns 2 and 3 contain the Sxl-binding poly-U stretches, whose localization in intron 2 varies but in intron 3 is conserved. The Sxl protein is fully conserved at the amino acid level in both species.

Amino Acid Sequence↗

PubMed1996

Protein-RNA and protein-protein interactions of the Drosophila sex-lethal mediated by its RNA-binding domains.

The Drosophila Sex-lethal (Sxl) contains two RNA-binding domains (RBDs) which belong to the RNA recognition motif (RRM) group. Sxl binds to a specific uridine-rich sequence which is believed to be the major cis-acting element for the splicing regulation of the transformer (tra) mRNA precursor. Here we show evidence supporting the previous suggestion that Sxl recognizes the sequence context downstream of the uridine-rich sequence. In addition, by means of UV-crosslinking assays with Sxl deletion constructs, we have demonstrated that Sxl RNA binding requires both of its RBDs for specificity and strength. Moreover, by the yeast two-hybrid analysis, we found that homodimeric interaction occurs between two Sxl molecules. Interestingly, the amino- and carboxy-terminal regions outside of the Sxl RBDs are dispensable for such dimerization, indicating that the protein-protein interaction is also mediated by RBDs. Coprecipitation experiments in vitro showed that the protein-protein interaction seems to be RNA-dependent but greatly enhanced by addition of the specific RNA containing the Sxl binding site, suggesting that the conformational change which is induced on binding to RNA may facilitate the interaction between Sxl molecules.

Animals↗

PubMed1995

EDA-containing fibronectin is synthesized from rheumatoid synovial fibroblast-like cells.

OBJECTIVE: To identify the cells that synthesize EDA-containing fibronectin (FN) and examine the role of EDA+FN in the pathogenesis of rheumatoid joint lesions. METHODS: Localization of EDA+FN and c-Fos protein in rheumatoid joints was studied immunohistochemically by utilizing antibodies for EDA+FN and c-Fos. Expression of EDA+FN was studied by immunoelectron microscopy and in situ hybridization. The amount of EDA+FN was measured by enzyme-linked immunosorbent assay. RESULTS: EDA+FN was specifically localized in the synovial lining layer of synovium with active rheumatoid arthritis (RA) (n = 17), but not in that with osteoarthritis (n = 4) or with inactive fibrous RA (n = 2). EDA+FN messenger RNA was localized in the synovial lining layer. EDA+FN was immunoelectron microscopically localized in the synovial lining fibroblast-like (type B) cells. EDA+FN was also detected at the cartilage-pannus junction and on the surface of RA cartilage. Double staining showed that EDA+FN colocalized with c-Fos protein in the rheumatoid synovial lining layer. Quantification of EDA+FN showed that it was highly concentrated in rheumatoid synovial fluids. CONCLUSION: EDA+FN is synthesized by the synovial lining fibroblast-like (type B) cells in situ in rheumatoid synovium, and appears to be expressed in association with activated or transformed states of synovium.

Animals↗

PubMed1994

Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection.

The Drosophila sex-lethal (Sxl) protein, a regulator of somatic sexual differentiation, is an RNA binding protein with two potential RNA recognition motifs (RRMs). It is thought to exert its function on splicing by binding to specific RNA sequences within Sxl and transformer (tra) pre-mRNAs. To examine the Sxl RNA binding specificity in detail, we performed in vitro selection and amplification of ligand RNAs from a random sequence pool on the basis of affinity with Sxl protein. After three cycles of selection and amplification, we cloned and sequenced 17 cDNAs corresponding to the RNAs selected in vitro. Sequencing showed that most of the RNAs selected contain polyuridine stretches surrounded by purine residues. In vitro binding analysis revealed that the sequences of the in vitro selected RNAs with relatively high affinity for Sxl show similarity to that of the Sxl- and tra-regulated acceptor regions, including the invariant AG sequence for splicing. These results suggest that Sxl recognizes and preferentially binds to a polyuridine stretch with a downstream AG sequence.

Animals↗

PubMed1994

Reduction of EDA(+) fibronectin and its clinical importance on cryofiltration.

Cryofiltration (CRYO) removes cryogel, which is a combination of fibrinogen (Fbg) and fibronectin (FN), containing pathological substances. The purpose of this study was to measure cryogel EDA(+) FN and study the relationship between EDA(+) FN and clinical symptoms in patients with rheumatoid arthritis, SLE and polymyositis. Cryogel contains 51 times more EDA(+) FN than plasma. The patients with rheumatoid arthritis showed a high level of EDA(+) FN in their plasma, and the EDA(+) FN level in plasma corresponded with changes in joint pain. We calculated the clearance level at several points in cryofiltration, and the reduction enabled us to evaluate the CRYO device. The EDA(+) FN clearance was 23.3 +/- 6.4 ml/min, the pFN clearance 16.5 +/- 4.1 ml/min, and the Fbg clearance 22.9 +/- 5.7 ml/min. As the plasma flow in cryofiltration was 30 ml/min, a clearance of EDA(+) FN and Fbg, approximately 23 ml/min, was obviously high. The study of the plasma level change of EDA(+) FN during cryofiltration revealed a temporary elevation. These results suggest that the EDA(+) FN was most efficiently reduced by cryofiltration, would become a good indicator on plasmapheresis, and might move from other tissues into the blood during cryofiltration.

Adolescent↗

PubMed1993

Cellular fibronectin in plasma: its implications in fibrinogen-associated cryoprecipitation and other related reactions.

Elimination of cryoprecipitable plasma components (cryogels) by cryofiltration from the circulating blood of patients with drug-resistant rheumatoid arthritis (RA) alleviates clinical symptoms including morning stiffness and arthralgia. The cryogels thus isolated from the blood were found to consist mainly of fibrinogen (Fbg) and fibronectin (FN). Analysis by immunoblotting with an anti-cellular FN monoclonal antibody revealed that cellular FN (cFN) co-existed with plasma FN (pFN) in the cryogels derived from the patients. Using an ELISA, we assessed cFN together with the total FN (pFN+cFN) in plasmas and cryogels derived from the patients. The cFN/total FN ratio was distinctly higher in the cryogels than in the plasmas, suggesting that cFN was more readily precipitated than pFN in association with Fbg under cold conditions.

Arthritis, Rheumatoid↗