Protein phosphatase Dusp26 associates with KIF3 motor and promotes N-cadherin-mediated cell-cell adhesion.

Recent studies have demonstrated essential functions for KIF3, a microtubule-directed protein motor, in subcellular transport of several cancer-related proteins, including the beta-catenin-cadherin(s) complex. In this study, we report identification of the protein-phosphatase Dusp26 as a novel regulator of the KIF3 motor. Here we undertake yeast two-hybrid screening and identify Kif3a, a motor subunit of the KIF3 heterotrimeric complex, as a novel Dusp26-binding protein. Co-immunoprecipitation and colocalization experiments revealed that Dusp26 associates not only with Kif3a, but also with Kap3, another subunit of the KIF3 complex. Dephosphorylation experiments in vitro and analysis using mutant forms of Dusp26 in intact cells strongly suggested that Dusp26 is recruited to the KIF3 motor mainly by interaction with Kif3a, and thereby dephosphorylates Kap3. Forced expression of Dusp26, but not its catalytically inactive mutant, promoted distribution of beta-catenin/N-cadherin, an established KIF3 cargo, to cell-cell junction sites, resulting in increased cell-cell adhesiveness. We also showed that Dusp26 mRNA expression was downregulated in human glioblastoma samples. These results suggest previously unidentified functions of Dusp26 in intracellular transport and cell-cell adhesion. Downregulation of Dusp26 may contribute to malignant phenotypes of glioma.

Primary erythrocytosis in a Japanese black calf: a case report.

An 8-month-old Japanese Black heifer with severe erythropoietic symptoms was subjected to clinical, histological and cytological examinations. During the 1 month clinical observation period, severe increases in RBC count, packed cell volume and haemoglobin concentration were observed. The plasma erythropoietin (Epo) concentration of the heifer (20.7 mIU/ml) was similar to that observed in normal control heifers. Blood gas examinations of the arterial and venous blood revealed low levels of partial pressure O(2) (PaO(2)), partial pressure CO(2) (PaCO(2)) and O(2) saturation (SaO(2)), while the blood pH was within the normal range. Gross lesions could not be detected. However, microscopic observation revealed severe proliferation of erythroblasts in the bone marrow and in the spleen without evidence of neoplastic changes. Based on these clinical and pathological examinations, we diagnosed the heifer as being the first case of primary erythrocytosis in Japanese Black cattle.

The mechanism of cell death in human cultured colon adenocarcinoma cell line COLO 201 induced by beta-D-N-acetylglucosaminyl-p-nitrophenol.

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-beta-N-acetylglucosamine (beta-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of beta-GlcNAc-PNP and 4-methylumbelliferyl-beta-N-acetylglucosamine, then the number of living cells was measured in a time course. In beta-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from beta-GlcNAc-PNP induced the cell death. Activity of beta-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

[Female carrier of Duchenne muscular dystrophy presenting with secondary dilated cardiomyopathy: a case report].

A 48-year-old female carrier of Duchenne muscular dystrophy had developed congestive heart failure but had no skeletal muscle symptoms. She was admitted to our hospital complaining of palpitation in December 1998. Her three sons had Duchenne muscular dystrophy. Neurological examination was unremarkable with no evidence of muscle weakness. Serum creatine kinase level was slightly increased. Echocardiography showed severe left ventricular dysfunction. Coronary angiography showed no abnormalities. Left ventriculography showed generalized hypokinesis and left ventricular ejection fraction was 28%. Dystrophin immunostaining of the skeletal muscle biopsy specimen showed a mosaic pattern. The dystrophin negative fibers were scattered among positive fibers. Cardiomyopathy is the only clinical manifestation of dystrophin gene mutation in carriers. Beta-blocker therapy(carvedilol 5 mg/day) was effective in this patient.

Pedunculated basal cell epithelioma which is not Pinkus tumor.

A 33-year-old female with pedunculated basal cell epithelioma was reported. She had noticed a cutaneous tumor on the scalp for two years before admission. It developed gradually and clinically resembled fibroma or pigmented nevus. Total resection was performed, and its histopathology revealed the solid or cystic type of basal cell epithelioma.

Effects of ATP on regulation of galactosyltransferase-I activity responsible for synthesis of the linkage region between the core protein and glycosaminoglycan chains of proteoglycans.

We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-beta-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 x g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100,000 x g centrifugation. When both fractions (the 100,000 x g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100,000 x g supernatant. In addition, the 100,000 x g precipitate, which was prepared from the 110 x g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100,000 x g supernatant activates galactosyltransferase-I activity.

Structural varieties of small proteoglycans in human spinal ligament.

Three types of small proteoglycan were purified from human spinal ligaments by ultracentrifugation, ion-exchange chromatography, gel-chromatography, and hydrophobic chromatography. Two of them were identified as decorin and biglycan, and the other was thought to be a decorin-subtype. Molecular sizes of decorin and decorin-subtype were both 85 kDa, and that of biglycan was 200 kDa. N-Terminal amino acid sequence of decorin-subtype corresponded with that of decorin, although it was different from decorin in terms of composition of amino acids and glycosaminoglycan chains, and reactivity with anti-human decorin antibody. The ratios of chondroitin sulfate to dermatan sulfate contained in the three proteoglycans were different, and the location of that in glycosaminoglycan chains was also thought to be different. It was demonstrated that three types of proteoglycan which are structurally different are present in extracellular matrix.

Acute toxicity of an anti-Fas antibody in mice.

By histopathologic examination of various organs in 3 normal strains, C3H/HeN, ICR, and DBA/1J, of mice treated intravenously once with anti-Fas antibody (Jo2), we failed to determine any target organ, except the liver, responsible for the acute lethality induced by the Fas/anti-Fas antibody interaction. However, we could show the presence of Fas-mediated apoptosis in other organs aside from the liver and normal mouse strain differences in susceptibility to anti-Fas antibody. Among these strains, C3H/HeN was the most susceptible to the antibody, followed by ICR and DBA/1J. We observed Fas-mediated apoptosis in the liver, spleen, thymus, lymph nodes, Peyer's patch, intestine, skin, coagulation glands, ovary, uterus, and vagina in all 3 strains and additionally in the epididymides and seminal vesicles in the DBA/1J strain. We also demonstrated that Fas-mediated apoptosis of small lymphocytes in the mantle zone of splenic lymphatic follicles preceded that of the hepatocytes or thymic cells. Since cellular damage was most severe in the liver among all the apoptotic organs in the 3 mouse strains, liver injury induced by anti-Fas antibody is speculated to play a significant role in the death.

Regulation of hyaluronate metabolism by progesterone in cultured fibroblasts from the human uterine cervix.

The effects of progesterone, dehydroepiandrosterone sulfate and 17beta-estradiol on the synthesis and degradation of hyaluronate were investigated using human uterine cervix fibroblasts. The cells were incubated with [3H]glucosamine in the presence of the hormones and then [3H]hyaluronate was isolated from the medium. The changes in the radioactivity of [3H]hyaluronate showed that progesterone suppressed hyaluronate synthesis by 22% of the control levels, while dehydroepiandrosterone sulfate and 17beta-estradiol enhanced it by 22% and 12% of the control levels, respectively. Furthermore, progesterone induced degradation of high-molecular-weight [14C]hyaluronate into low-molecular-weight hyaluronate (Mr approximately 40000). These results suggest that in cultured fibroblasts from the human uterine cervix progesterone converts hyaluronate metabolism from the synthesis phase to the degradation phase.

Characterization of mucin in whole-gut lavage fluid obtained from patients with inflammatory bowel disease.

Whole-gut lavage fluid, collected by administering an electrolyte lavage solution orally, was found to be an excellent and easily collectable source of abundant mucin. Furthermore, the biochemical features of the mucin from patients with ulcerative colitis and Crohn's disease were investigated. The mucin was separated into four fractions by Sepharose CL-4B, Sepharose CL-2B, and DEAE Sephacel chromatography. Compared with healthy subjects, the total yields of mucin from ulcerative colitis patients were low due to a deficiency of neutral mucin, whereas those from Crohn's disease patients were high, which was attributable mainly to high-molecular-weight mucin. The fucose and sulfate contents were low in ulcerative colitis, but only the former was low in Crohn's disease. The different biochemical features of the mucin obtained from whole gut lavage fluid appear to reflect mucosal pathological changes associated with inflammatory bowel disease.

Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix.

High-molecular-weight [14C]hyaluronate was incubated with cultured fibroblasts from human uterine cervix and skin, and then the depolymerization of the hyaluronate was investigated. [14C]Hyaluronate in the medium of skin fibroblasts was depolymerized into a constant molecular weight (M(r) about 40,000), whereas that of cervix fibroblasts was not depolymerized, irrespective of incubation period. However, when progesterone was added to the medium of cervix fibroblasts, hyaluronate was depolymerized to the same extent as that in skin fibroblasts. The reducing terminal sugar of the depolymerized hyaluronate was N-acetylglucosamine. These results suggest that a hyaluronate-depolymerizing enzyme, endo-beta-N-acetylglucosaminidase, was induced by progesterone in cultured fibroblasts derived from human uterine cervix.

Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin).

The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N-terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets.

Complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein and its homology to histocompatibility antigens.

In the present study the complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein was determined. This protein whose biological function is unknown consists of a single polypeptide chain of 276 amino acid residues including 8 tryptophan residues and has a pyroglutamyl residue at the amino terminus. The location of the two disulfide bonds in the polypeptide chain was also established. The three glycans, whose structure was elucidated with the aid of 500 MHz 1H NMR spectroscopy, were sialylated N-biantennas. The molecular weight calculated from the polypeptide and carbohydrate structure is 38,478, which is close to the reported value of approximately equal to 41,000 based on physicochemical measurements. The predicted secondary structure appeared to be comprised of 23% alpha-helix, 27% beta-sheet, and 22% beta-turns. The three N-glycans were found to be located in beta-turn regions. An unexpected finding was made by computer analysis of the sequence data; this revealed that Zn-alpha 2-glycoprotein is closely related to antigens of the major histocompatibility complex in amino acid sequence and in domain structure. There was an unusually high degree of sequence homology with the alpha chains of class I histocompatibility antigens. Moreover, this plasma protein was shown to be a member of the immunoglobulin gene superfamily. Zn-alpha 2-glycoprotein appears to be a truncated secretory major histocompatibility complex-related molecule, and it may have a role in the expression of the immune response.

Chemical characterization of urinary glycopeptides.

We prepared human urinary glycopeptides from the supernatant liquid remaining after precipitation of the nondialyzable fraction with cetylpyridinium chloride. Using cation exchange and affinity chromatographies and gel filtration, we obtained 28 glycopeptide subfractions. By compositional analyses of sugar and amino acid, and by reducing-terminal analyses after reduction with NaBH4, we determined the size of the carbohydrate moiety and the types of carbohydrate-peptide linkage involved. We isolated several glycopeptides not previously described: six with sialic acid, two with fucose, two with glucose, and one with N-acetylgalactosamine. The sialic acid glycopeptides had a short carbohydrate chain of the O-glycoside type. The fucose-containing glycopeptides were fucosyllactosaminoglycans. The glucose glycopeptides were polymers linked to a small peptide moiety. The N-acetylgalactosamine-rich glycopeptide was found in an N-glycoside-type fraction, with N-acetylgalactosamine at the nonreducing terminal.

Erythropoietic protoporphyria--report of a family and clinical review.

Three cases of erythropoietic protoporphyria are reviewed. The three patients constitute members of one family. The protoporphyrin content of the red blood cells was high, but porphyrins and their precursors in the urine and faeces were not excessive. Other normal members of the family did not reveal high protoporphyrin content in the red blood cells. Clinical symptoms were itching, swelling, shallow depressed scars and waxy yellow discoloration on the face and brown pigmentation and thickness on the back of the hands after exposure to the sun. The microscopic findings from skin biopsy specimens of the lesions resembled changes of the lipoid proteinosis.