Variants deficient in phagocytosis of latex beads isolated from the murine macrophagelike cell line J774.

Variants that lack the ability to ingest latex beads were isolated from the mouse macrophagelike cell line J774. Carboxylated latex beads were derivatized with polylysine and then daunomycin by a carbodiimide method. Cells that ingested such beads were killed; variants that survived were isolated. Variants were detected at very low frequency and only after nitrosoguanidine mutagenesis. Of 11 independent isolates, 10 showed a lowered rate of uptake of polystyrene beads (without daunomycin). All of these proved normal in rate and extent of Fc-mediated phagocytosis. There was essentially no change in sensitivity to free daunomycin in the variants compared to the parent. These results support the previous hypothesis that there are differences in the metabolic routes of receptor-mediated and nonspecific phagocytosis.

Increase of sialylated tetraantennary sugar chains in parallel to the higher lung-colonizing abilities of mouse melanoma clones.

The N-linked sugar chains of melanoma cell membrane from five murine B16 melanoma clones (F1, F10, BL6, W1-4, and C4-1) with different degrees of metastatic abilities after intravenous and intrafootpad injections were released quantitatively as oligosaccharides by hydrazinolysis, and their structures were analyzed by serial lectin column chromatography, Bio-Gel P-4 column chromatography, and sequential glycosidase digestion. Sugar chain structures of each clone have shown to consist of the same elemental oligosaccharides, but to differ in their percent compositions. More than 84% of the neutral oligosaccharides were high mannose-type sugar chains. Most complex-type sugar chains were sialylated, of which the major structure was tetraantennary sugar chain. Highly lung-colonizing F10 cells had 1.4 and 1.7 times more non-repeated tetraantennary sugar chains than moderately colonizing F1 and C4-1 cells, respectively, and 2.5 times more than poorly colonizing W1-4 cells. BL6 cells, which are also highly lung-colonizing, had 1.5 and 1.9 times more non-repeated tetraantennary sugar chains than F1 and C4-1 cells, respectively, and 2.8 times more than W1-4 cells. These results suggest that increase of sialylated tetraantennary complex-type sugar chains without N-acetyllactosamine repeating units of B16 melanoma cells might correlate with the higher lung-colonizing ability after intravenous injection.

O-Glycosylation of G-protein-coupled receptor, octopus rhodopsin. Direct analysis by FAB mass spectrometry.

In addition to the N-glycan that is evidently conserved in G-protein-coupled receptors (GPCRs), O-glycans in the N-terminus of GPCRs have been suggested. Using a combination of enzymatic and manual Edman degradation in conjunction with G-protein coupled receptor mass spectrometry, the structure and sites of O-glycans in octopus rhodopsin are determined. Two N-acetylgalactosamine residues are O-linked to Thr4 and Thr5 in the N-terminus of octopus rhodopsin. Further, we found chicken iodopsin, but not bovine rhodopsin, contains N-acetylgalactosamine. This is the first direct evidence to determine the structure and sites of O-glycans in GPCRs.

Role of terminal galactose residues in N-linked sugar chains of sheep erythrocyte membrane glycoproteins in rosette formation with T lymphocytes.

In this study, we found that rosette formation of T lymphoblastic Molt-3 cells with sheep erythrocytes is inhibited by addition of membrane glycoproteins which were solubilized from sheep erythrocyte ghosts by the lithium diiodosalicylate extraction methods. Their rosetting inhibitory activity was markedly reduced by digestion with N-glycanase, but not with O-glycanase. The inhibitory activity was also reduced by beta-galactosidase digestion, while it was enhanced by desialylation. These observations indicate that nonreducing terminal galactose residues of N-linked sugar chains included in membrane glycoproteins on sheep erythrocytes are important for rosette formation with T lymphocytes.

Molecular and immunological approaches to mammalian fertilization.

By means of hybridoma technology, we obtained six hydriboma cell lines producing monoclonal antibody (mAb) to porcine zona pellucid (ZP), two of which recognizes the steric structure of common antigens between porcine ZP and humans. Furthermore, we have analyzed all or partial structures of N- and O-linked sugar chains of ZP glycprotein from porcine or murine oocytes. Then, we have clarified that beta-galactose and Le(X) residues on ZP played the binding roles to sperm cells in porcine and murine fertilization. We have also succeeded Sp38 cDNA cloning from cDNA library of porcine testis. We found that Sp38 protein bind to porcine ZP2 and expressed in murine and human sperm cells. Corresponding to the presence of major histocompatibility complex (MHC) class II on murine sperm, CD4 on the murine egg plasma membrane was clearly shown by indirect IIF and immunoprecipitation test. Furthermore, the transcriptional expression of CD4/p56(lck) in eggs was confirmed by RT-PCR method. In addition, the p56(lck) associated with CD4 underneath the plasma membrane of eggs was autophosphorylated after cross-linking of CD4 with anti CD4 mAb. The binding between eggs or Sf9-CD4 cells labeled with anti-CD4 mAb and sperm cells labeled with anti-monomorphic region of class II mAb was completely blocked. Considering these findings together with the fact that an interspecies' heterogeneity is present in CD4 amino acid sequence at the interactive site with class II, we elucidated that one of species' specific intercellular adhesions between two gametes at the fusion step in fertilization is definitely mediated by class II located on the posterior region of sperm head and CD4/p56(lck) complex on the plasma membrane of egg.

Purification and structural analysis of extracellular matrix of a skin tumor from a patient with juvenile hyaline fibromatosis.

Juvenile hyaline fibromatosis is a rare mesenchymal dysplasia that is inherited in an autosomal recessive fashion. The histological features of the tumor-like lesions are characterized by the deposition of amorphous hyaline material in the extracellular spaces of the dermis and soft tissues. We have analyzed the hyaline substance in a specimen of a skin tumor obtained from a 4-year-old Japanese girl with juvenile hyaline fibromatosis. It was found to consist mainly of type VI collagen; a small amount of type I collagen was also present. These components were separated by DEAE-cellulose ion-exchange chromatography under reducing conditions. The ratio of the dry weights of type I and type VI collagen was 1:4. Of the three chains of type VI collagen (alpha 1(VI), alpha 2(VI) and alpha 3(VI)), alpha 3(VI) was the most abundant. Glycosaminoglycans in the tumor tissue comprised dermatan sulfate, chondroitin sulfate and hyaluronan, with dermatan sulfate predominating. In contrast, hyaluronan is the most abundant in normal skin.

The asparagine-linked sugar chains of the glycoproteins in calf thymocyte plasma membrane. Structural studies of neutral oligosaccharides.

The neutral oligosaccharide fraction obtained from the hydrazinolysate of the plasma membrane glycoproteins of calf thymocytes was shown to be a mixture of twelve oligosaccharides. Oligosaccharides A, B, and C, which released galactose on treatment with jack bean beta-galactosidase but not with diplococcal beta-galactosidase, were shown to have the structures Gal beta 1-3Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-6(3)[Gal beta 1-3Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3(6)]Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc, Gal beta 1-3Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-6(3)[Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3(6)]Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc, and Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc, respectively. Oligo saccharides D and E, which released galactose on treatment with either jack bean beta-galactosidase or diplococcal beta-galactosidase, were shown to have the structures Gal beta 1-4GlcNAc beta 1-2Man alpha 1-(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-)GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-)GlcNAc, respectively. Five oligosaccharides (F1, G, H1, I, and J) were shown to have typical high-mannose type structures (Man alpha 1-)n.Man beta 1-4GlcNAc beta 1-GlcNAc, with n-values of 8, 7, 6, 5, and 4, respectively. The structures of the remaining two oligosaccharides F2 and H2 were elucidated as GlcNAc beta 1-2Man alpha 1-(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-)GlcNAc and Man alpha 1-(Man alpha 1-)Man beta 1-(GlcNAc beta 1-Man alpha 1-)Man beta 1-GlcNAc beta 1-GlcNAc, respectively.

Asparagine-linked sugar chains of glycoproteins in calf thymocyte plasma membrane. Isolation and fractionation of oligosaccharides liberated by hydrazinolysis.

The plasma membrane glycoproteins of calf thymocytes were converted to glycopeptides by exhaustive pronase digestion. Glycopeptides with asparagine-linked sugar chains were separated from those with mucine-type sugar chains by Bio-Gel P-10 column chromatography. The asparagine-linked sugar chains were released as oligosaccharides from the peptide moiety by hydrazinolysis and labeled by reduction with NaB[3H]4. The radioactive oligosaccharides were fractionated into fifteen acidic components and ten neutral components by combination of paper electrophoresis and Bio-Gel P-4 column chromatography. The acidic nature of all fifteen acidic components can be ascribed to their N-acetylneuraminic acid residues. The Bio-Gel P-4 column chromatographic patterns of the neutral oligosaccharide fraction and of the neutral fraction obtained on sialidase treatment of the pooled acidic oligosaccharide fraction were totally different, indicating that the acidic oligosaccharides are not simple sialyl derivatives of the neutral oligosaccharides.

Structural study of the sugar chains of human lactoferrin: finding of four novel complex-type asparagine-linked sugar chains.

Human lactoferrin contains 2 asparagine-linked sugar chains in 1 molecule. These sugar chains were released as oligosaccharides by hydrazinolysis from two lactoferrin samples of different races. The two oligosaccharide fractions gave exactly the same fractionation pattern upon paper electrophoresis and Bio-Gel P-4 column chromatography after sialidase digestion. A structural study of the oligosaccharides obtained from the two samples by sequential exoglycosidase digestion in combination with methylation analysis also gave the same results indicating that there is no racial difference both in quality and quantity of the sugar chain moiety of lactoferrin. In addition to the two acidic sugar chains, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlucNAc and Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 1Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc, and two novel acidic sugar chains, Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2 Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6(GlcNAc and Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc, were found to occur in human lactoferrin.

Structural studies of the sugar chains of cold-insoluble globulin isolated from human plasma.

The asparagine-linked sugar chains of cold-insoluble globulin isolated from human plasma were released as oligosaccharides from the polypeptide moiety by hydrazinolysis. These oligosaccharides were N-acetylated and could be labeled by reduction with NaB[3H]4. The yield of radioactive oligosaccharides indicated that the glycoprotein has four asparagine-linked sugar chains in one molecule. More than 90% of the radioactive oligosaccharides contain N-acetylneuraminic acid, and could be separated into two acidic oligosaccharides, A-1 and A-2. By sequential exoglycosidase digestion in combination with methylation studies, their structures were elucidated as Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3) Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 (NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2 Man alpha 1 leads to 3)-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.

Comparison of the catalytic properties of thrombin and trypsin by kinetic analysis on the basis of active enzyme concentration.

The interaction of bovine thrombin [EC 3.4.21.5] with synthetic substrates and products was studied. The enzyme was purified from Parke-Davis topical thrombin. The purification process afforded some preparations with different clottin specific activities but with similar esterase specific activities. The preparation having highest clotting specific activity and that having lowest clotting activity were tentatively named thrombin-C and thrombin-E, respectively. Kinetic parameters for the hydrolysis of synthetic substrates and normality titrants were determined on the basis of active enzyme quantity, which was assayed by means of a fluorometric normality titrant. It was shown that thrombin-E was acylated by the substrates more slowly than thrombin-C, while deacylation proceeded at similar rates in the two preparations. The results were also compared with those obtained with bovine trypsin [EC 3.4.21.4]. The acylation rates of both thrombin preparations were markedly lower than that of trypsin, while the deacylation rates of the former were only slightly lower than that of the latter. The effects of various product-type inhibitors, such as benzyloxycarbonyl-, benzoyl-, and tosyl-L-arginine, were also examined. Thrombin was affected by these inhibitors not competitively, though trypsin was inhibited competitively.

Structure of asparagine-linked sugar chains in the major polypeptide of hepatitis B surface antigen.

Asparagine-linked sugar chains were quantitatively released from hepatitis B surface antigen on hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The released oligosaccharides were composed of two major sialylated components and a trace of a neutral component. From the results of the combination of sequential exoglycosidase digestion and methylation analysis, the structures of the acidic and the neutral components were deduced to be NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3(+/- NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAcOT and Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAcOT, respectively.

Human type VI collagen: purification from human subcutaneous fat tissue and an immunohistochemical study of morphea and systemic sclerosis.

Collagen was isolated from human placenta by pepsin digestion and salt precipitation. This collagen was similar in its electrophoretic mobility and immunological reactivity with monoclonal antibody to form B of type VI collagen in the literature (Trueb B, Schreier T, Bruckner P and Winterhalter K. 1987. Eur. J. Biochem. 166: 699-703). We prepared polyclonal rabbit antiserum against alpha 2 chain of type VI collagen and performed an immunohistochemical study using this polyclonal antibody. It reacted in fat tissue and around vessels and peripheral nerves in normal human skin. To confirm the presence of type VI collagen in fat tissue, we isolated collagen from human subcutaneous tissue. This collagen showed a similar pattern in polyacrylamide gel electrophoresis with that from human placenta and cross-reacted with monoclonal or polyclonal antibody against type VI collagen. By immunohistochemical staining, abundant type VI collagen was observed in the septum of subcutaneous fat tissue in morphea or systemic sclerosis. In the mild hyalinizing areas or after treatment with 6M urea or hyaluronidase in highly hyalinized areas, the staining of type VI collagen increased. These data suggest that the amount of type VI collagen in subcutaneous tissue is involved in the early phases of these fibrosing disorders and that type VI collagen accumulates even more in hyalinizing tissue in late phases of these diseases.

Postoperative hyponatremia in a patient with ACTH-producing Merkel cell carcinoma.

Merkel cell carcinoma is characterized by specific neuroendocrine features and the expression of several neuropeptides. We report a case of Merkel cell carcinoma with post-surgical hyponatremia in an 85-year-old Japanese woman. A tumor on the left cheek histopathologically showed the characteristics of Merkel cell carcinoma together with Bowen's disease. Although an increased level of ACTH was found both in the tumor and in the peripheral blood, the postoperative hyponatremia in our patient seems more likely to have been caused by the stress of the operation and indapamide, considering that the ACTH level in the tumor was much lower than those in other ectopic ACTH-producing tumors in previous reports.

A case of Goltz syndrome presenting as congenital incomplete alopecia.

A 5-year-old Japanese girl had had localized, incomplete hair loss on the scalp, minimal distichia, and a small papillomatous eruption on the right upper eyelid since birth. The diagnosis of Goltz syndrome was made by histological findings such as upward extension of the subcutaneous tissue to the papillary dermis and marked diminution in the thickness of the dermis, although typical linear atrophy-like eruptions and other mesoectodermal dysplasia were absent.

Squamous cell carcinoma in a renal transplant recipient with linear porokeratosis.

A 40-year-old man developed squamous cell carcinoma on a perianal lesion of linear porokeratosis after renal transplantation. The tumor metastasized to the left inguinal lymph node 25 months after the primary tumor was excised. p53 overexpression was observed in the tumor cells, but not in the porokeratotic lesion. Interestingly, continuous subcutaneous infusion of peplomycin for the lymph node metastasis significantly improved the warty lesions of porokeratosis. In this patient, immunosuppressive agents might have accelerated the development of carcinoma on a skin area with malignant potential.

Gas gangrene following sacral pressure sores.

We report two cases of gas gangrene developed from sacral pressure sores. The first case was clostridial and the second, non-clostridial gas gangrene. Both patients died within two months. The first patient, a 56-year-old woman suffering from palsy of the lower half of the body for 3 weeks, developed a sacral pressure sore. One month later, crepitus by palpation and gas formation in the X-ray film were detected in the hip and right thigh. A culture of odoriferous pus yielded Clostridium limosum in addition to Staphylococcus intermedius, Enterococcus faecalis, Pseudomonas aeruginosa, and Bacteroides fragilis. Blood culture yielded Bacteroides fragilis. The patient died 50 days after admission in spite of surgical debridement and aggressive therapy with high doses of antibiotics and hyperbaric oxygen. The second patient, a 70-year-old man suffering from diabetic nephropathy, arteriosclerosis obliterans of the lower limbs, and cerebral infarction, developed a large decubitus ulcer covering the whole sacral area. Crepitus and gas were detected in the soft tissue of the left gluteal region. Almost the entire gluteus maximus muscle was necrotic. Bacteroides fragilis, methicillin-resistant or -sensitive Staphylococcus aureus and Escherichia coli were isolated from the muscle. Bacteroides fragilis was also obtained by blood culture. The patient died on the 72nd day after admission.

[Magnetic resonance imaging (MRI) of bone marrow necrosis].

Magnetic resonance (MR) of bone marrow was studied in two cases of acute leukemia which showed bone marrow necrosis. Case 1:A 24-year-old female was admitted because of sternum pain and bleeding tendency. She was diagnosed AML based on the peripheral blood picture. Bone marrow biopsy revealed the presence of bone marrow necrosis. T1 weighted imaging of MR showed low signal intensity in all vertebral marrow. Fatty marrow was demonstrated after achieving complete remission and the MR imaging of bone marrow changed to show high intensity, suggesting fat deposition. Case 2: A 19-year-old female suffered from chest pain and lumbago, and was diagnosed as ALL. DIC and bone marrow necrosis were confirmed during chemotherapy for remission induction. T1 weighted imaging showed the mosaic pattern of low and high signal intensity. She achieved complete remission and bone marrow clot revealed the presence of fatty marrow. Most areas of low signal intensity of T1 weighted imaging changed to those of high signal intensity. These observations suggest that necrotized bone marrow seemed to change to fatty marrow along with achieving remission. MR imaging study of bone marrow is useful for evaluating hematopoiesis in hematologic disorders.

[Local chemotherapy by a sustained-release preparation with fibrin seal against the operative wound in head and neck cancer].

Fibrin seal has been used for hemostasis and sealing in operative field of tumors in the head and neck. The authors applied it for drug preparation and tried a local chemotherapy to treat residual and disseminated tumors of cellular level in the operative wound using 5-FU. The drug release rate in this therapy in vitro study was 50% after 24 hrs. When injected to rats bearing Yoshida sarcoma, it exhibited a marked antitumor effect compared to the control group given 5-FU alone. This therapy is easy to make the dosage adjustment and can apply drugs directly to the tumor residue at the high concentration. It will be clinically a useful adjuvant therapy for radiotherapy, surgery or chemotherapy.

[A case of colon cancer with local recurrence successfully treated by systemic and local intra-arterial chemotherapy using a combination of 5-fluorouracil, interferon-alpha 2A, leucovorin and carboplatin].

A 59-year-old man with colon cancer was diagnosed as having a local recurrence of the disease, forming a huge intra pelvic tumor, accompanied by pulmonary metastasis 16 months after hemicolectomy. He received alternate systemic and local chemotherapy consisting respectively of a 5-day course of continuous infusion of 5-FU 600 mg/m2/day, bolus injection of leucovorin (LV) 20 mg/m2/day, intramuscular injection of interferon (IFN)-alpha 2a 6 x 10(6) IU/day, and intra-arterial administration of 5-FU, LV and carboplatin using reservoir catheter through pudendal artery, each repeated every 3 weeks. After 6 systemic and 8 local treatments, metastatic lesions disappeared and the intra-pelvic tumor shrunk by 62%, indicating a partial response. The patient then underwent dissection of the intra-pelvic tumor. Pathological examination indicated a curative resection. Alternate systemic and local intra arterial chemotherapy using a combination of 5-FU, LV, IFN and and carboplatin is highly promising for metastatic colorectal cancer with local recurrence.