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Biomedical subjects

M Fukuzawa

Publications and source records attributed to M Fukuzawa.

At least 109 records · Page 6Linked to original sources
PubMed1994

Cycling of an 85-kDa lysosomal membrane glycoprotein between the cell surface and lysosomes in cultured rat hepatocytes.

We studied the endocytic transport of an 85-kDa lysosomal membrane glycoprotein (LGP85) from the cell surface to lysosomes in cultured rat hepatocytes. Fab' fragments of a monoclonal antibody against LGP85 (YA30 mAb) were conjugated with horseradish peroxidase (HRP) and then used as probes to monitor the endocytic transport of LGP85 from the plasma membrane to lysosomes. Continuous internalization and lysosomal transport of HRP-YA30 mAb Fab' occurred in the hepatic cells, resulting in its accumulation in the dense lysosomal fraction obtained from the cells on Percoll density centrifugation. The endocytic transport of HRP-YA30 mAb continued in the presence of the protein synthesis inhibitor, cycloheximide, indicating that LGP85 is cycled between the cell surface and lysosomes or endosomes, like other lysosomal membrane glycoproteins, lamp-1 and lamp-2, as reported previously [Akasaki et al. (1993) J. Biochem. 114, 598-604]. The half times (t1/2) of internalization and lysosomal transport of LGP85 were 32 min and 2.0 h, respectively. The kinetics of endocytic transport for LGP85 are very similar to those of lamp-1 and lamp-2. LGP85 possesses a short cytoplasmic tail whose amino acid sequence is quite different from those of lamp-1 and lamp-2. Therefore, these results suggested that continuous internalization from the cell surface and lysosomal transport of of endogenous LGP85 occur through a mechanism that can recognize this novel amino acid sequence, probably a Leu-Ile-containing motif, in normal hepatic cells of rat.

Amino Acid Sequence↗
PubMed1993

Different subcellular localizations of discoidin I monomer and tetramer in Dictyostelium discoideum cells: using conformation-specific monoclonal antibodies.

We characterized four monoclonal antibodies that recognize monomeric and/or tetrameric forms of Dictyostelium lectin (discoidin I) to study intracellular localization of this lectin in early development. Three different PAGEs (native-, urea-, and SDS-PAGE) following immunoblot showed that three of the four mAbs exhibit preference for the tetrameric form while mAb DC2 reacts only with unreduced monomer. By immunofluorescence studies of Dictyostelium NC-4, we found that the anti-tetramer antibodies mainly stain multilamellar bodies, which are food vacuoles to be externalized from cells. In contrast, mAb DC2 stains the cytosol weakly but not the multilamellar bodies. The same pattern of distribution was confirmed by immunoelectron microscopy. These results clearly indicate that, upon packaging discoidin I into multilamellar bodies, the tetrameric form is selectively packaged from the cytosolic pool. In addition, to clarify the relation between the multilamellar bodies and the tetramer, we examined the localization of tetramer using both the cells fed by Escherichia coli and the A3 cells axenically grown. When the cells were fed by E. coli, the cells made multilamellar bodies that contained no lectin; instead the tetramer was found in the cytoplasm and the cells still exclude the lectin around the cells. In contrast, A3 cells grown axenically without bacteria do not make multilamellar bodies but the tetramer also was found in the cytoplasm evenly (also, see W.F. Loomis, Dictyostelium Discoideum, pp. 160-161, Academic Press, New York, 1975). The data suggest that, under these conditions, the tetramer can be excluded from cells via some organelle such as normal secretory vacuole but not multilamellar bodies. Moreover this represents the first example of differential localization of tetramer and monomer of discoidin I in the cellular slime mold cells.

Animals↗
PubMed1993

Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of Cryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method. C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10(7) cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells of C. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure of C. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10-13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions by C. neoformans.

Animals↗
PubMed1993

Cycling of two endogenous lysosomal membrane proteins, lamp-2 and acid phosphatase, between the cell surface and lysosomes in cultured rat hepatocytes.

Our previous studies provided evidence that a 107-kDa major lysosomal membrane glycoprotein termed lamp-1 shuttles between lysosomes and the plasma membrane along the endocytic pathway in rat hepatic cells [Furuno et al. (1989) J. Biochem. 106, 708-716; Furuno et al. (1989) J. Biochem. 106, 717-722]. In the present study, we investigated the movement of a 96-kDa major lysosomal membrane glycoprotein, referred to as lamp-2, and lysosomal acid phosphatase (LAP) in the endocytic membrane transport system of cultured rat hepatocytes. Fab' fragments of anti-lamp-2 and anti-LAP antibodies conjugated with horseradish peroxidase (HRP) were used as probes to analyze quantitatively the transport of these two membrane proteins from the cell surface to lysosomes. After the addition of HRP-anti-lamp-2 and anti-LAP Fab' fragments to the culture medium, the delivery of the antibody conjugates to lysosomes was examined by cell fractionation on a Percoll density gradient. The amount of these HRP tracers in the lysosomal fraction became larger as the period of cell incubation was increased. Km values for uptake of HRP-anti-lamp-2, and LAP Fab' fragments were 0.74 and 0.62 microM, respectively, which were comparable to that of HRP-anti-lamp-1 Fab' (0.57 microM). The endocytic process of the two HRP-antibodies continued for an extended period in the cells exposed to the protein synthesis inhibitor, cycloheximide. Furthermore, we measured the transit times of HRP-anti-lamp-1, anti-lamp-2, and anti-LAP Fab' fragments from the cell surface to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1992

Massive small bowel resection in neonates--is weaning from parenteral nutrition the final goal?

Eight surviving cases from an original 15 pediatric patients who underwent massive small bowel resection during their neonatal period were reviewed for a period of 2-19 years to assess long-term prognosis. the primary diseases were congenital intestinal atresia in 6 cases and midgut volvulus in 2. The length of the residual small intestine ranged from 27-75 cm and the ileocaecal valve had been resected in 3 cases. All cases were able to be weaned from parenteral nutrition and at present, 6 of the children can tolerate normal meals but 2 are still receiving enteral nutrition, in the form of a low residue diet at home. Near normal somatic growth was achieved in the most recent 5 cases who received intensive nutritional treatment in the immediate postoperative period, whereas 3 who did not receive nutritional management exhibited growth retardation. The D-Xylose absorption test revealed gradual improvement and finally normal absorption in all except one case with the shortest remaining intestine (27 cm). However, the fat absorption test revealed abnormal absorption in cases with a residual intestine of less than 45 cm. Late metabolic complications such as renal calculi, cholelithiasis and pathologic fractures were encountered in 3 cases.

Child↗
PubMed1992

Anterior sagittal anorectoplasty for rectovestibular and anovestibular fistula.

Rectovestibular (or anovestibular) fistula is the most common form of anorectal anomaly in female infants. In the surgical repair of these malformations, anal transplantation has been in widespread conventional use. Based on the idea of posterior sagittal anorectoplasty, we devised a new approach, anterior sagittal anorectoplasty, in which, in the lithotomy position, the anterior portion of sphincter muscles are cut through a median perineal skin incision and then the rectum is pulled through the center of these muscles to make its opening at the normal position. The operation has been performed in 12 patients with satisfactory results.

Anal Canal↗
PubMed1992

Injection of bleomycin as a primary therapy of cystic lymphangioma.

Forty-five patients (19 boys and 26 girls) with lymphangioma have been treated at Osaka University Hospital during the 10-year period from January 1978 to December 1987. Twenty-nine underwent initial bleomycin (BLM) therapy. These patients' lesions consisted of large cystic masses that were demonstrated by echography or computed tomography scans. BLM was administered as a solution of 1 mg/mL in saline, after aspiration of the cyst contents using a puncture needle. The total dose ranged from 3 mg to 32 mg (average, 10 mg) and was administered between 1 and 16 times. Of the 29 cases treated with BLM, 25 cases (86%) showed significant reduction of the mass and 16 (55%) showed complete disappearance. Recurrence occurred in 3 patients. One responded to further injection of BLM, and the other 2 required surgical resection. Thirteen patients required surgical resection; however, in those cases, the previous injection of BLM was found to minimize the extent of surgical resection required. There were no serious side effects with BLM injection. Injection of BLM as an initial treatment of cystic lymphangioma was found to be very useful. It can reduce the size of the mass and even result in complete disappearance; consequently, it can minimize the frequency and extent of surgical intervention.

Bleomycin↗
PubMed1992

Isolation and characterization of a novel membrane glycoprotein of 85,000 molecular weight from rat liver lysosomes.

We have purified and characterized a novel glycoprotein (r-lamp-3) with an apparent molecular weight (Mr) of 85,000 from membranes of triton-filled lysosomes (tritosomes) by the use of immunoaffinity chromatography on a column of monoclonal antibody-Sepharose 4B. r-lamp-3 accounted for approximately 4% of the total proteins in tritosomal membranes. The isoelectric point (pI) of r-lamp-3 was 4.5 and it was shifted to 6.5 after neuraminidase treatment with its molecular weight decreased by about 7000. Pulse-chase experiments in cultured rat hepatocytes using [35S]methionine showed that r-lamp-3 was initially synthesized as a 77,000 polypeptide and processed to a mature protein with an Mr of 85,000. Upon treatment with endo-beta-N-acetylglucosaminidase H (Endo H), the precursor and mature forms were converted to 55,000 and 73,000 polypeptides, respectively. From the Mr reduction of the precursor form, we estimated the presence of 10--12 N-linked oligosaccharides/r-lamp-3 polypeptide. The data on enzymatic deglycosylation suggested that the mature form of r-lamp-3 contained the same numbers of high mannose-type and complex-type N-linked oligosaccharide chains.

Animals↗
PubMed1992

Epithelioid hemangioendothelioma of the liver diagnosed by liver biopsy under laparoscopy.

We report a case of primary epithelioid hemangioendothelioma arising in the liver. There were no specific findings or signs except for the deformity and multiple lesions of the liver at imaging procedures, which included magnetic resonance imaging. The key to the diagnosis was histological examination of liver biopsy specimens obtained under laparoscopy. Plasma factor VIII activity was elevated, which was also helpful in the diagnosis.

Biopsy↗
PubMed1990

Recovery of the contact site A glycoprotein of Dictyostelium discoideum from sodium dodecyl sulfate-polyacrylamide gel and characteristics of monoclonal antibodies against the recovered protein.

Monoclonal antibodies were produced against a cell-cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X-114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross-reacted heavily with other membrane glycoproteins.

Antibodies, Monoclonal↗