Swelling behavior of self-assembled monolayers of alkanethiol-terminated poly(ethylene glycol): a neutron reflectometry study.

The swelling behavior of alkanethiol-terminated poly(ethylene glycol) with an average molecular weight of 2180 Da (i.e., approximately 45 ethylene glycol, EG, units) in contact with water was investigated by neutron reflectometry as a function of the morphology of the PEG-SH layer. Amorphous films at a low grafting density show significant swelling with an increase of the film thickness from approximately 25 A in the dry state to approximately 70 A in contact with D2O, which corresponds to a total water uptake of approximately 38 mass %. In contrast, quasi-crystalline monolayers exhibit only a small amount of water penetrating into the film (approximately 8 mass %) with a corresponding change of the layer thickness from approximately 110 to approximately 125 A. The water uptake per EG unit corresponds to the literature value of 1.5 for the amorphous layer and to only 0.25 in the case of the quasi-crystalline film.

Molecular gradient along the axon pathway is not required for directional axon growth.

We show that axon guidance of embryonic hippocampal neurons is promoted by pathways of a decapeptide (RDIAEIIKDI) derived from a neurite outgrowth domain of the gamma1 chain of laminin-1. This guidance is directly dependent on: (1) a concentration difference of the decapeptide between the peptide pathway and its surrounding areas, and (2) the optimal surface geometry of the decapeptide pathway. These results indicate that axon guidance of central neurons may proceed along a preferred substratum pathway without a concentration gradient of the guiding molecule along this pathway, or without a repulsive molecule next to the axon pathway.

Monoclonal antibody against ependymoma-derived cell line.

Mouse myeloma cells were fused with spleen cells from mice that had been immunized with a human ependymoma derived cell line, KMS II. Hybridomas producing monoclonal antibodies (MAbs) were screened and cloned. Specificity of the antibody was determined by enzyme-linked immunosorbent assay (ELISA) and/or indirect immunofluorescence assay. One of the MAbs, designated Ep-C4 (subclass = IgG1), reacted with two cell lines derived from ependymoma but did not react with 17 cell lines derived from other types of brain tumor nor with 4 neuroblastoma cell lines or 19 cell lines derived from carcinoma, hematopoietic tumors and amnion. Indirect immunofluorescence and immuno-electron microscopy studies revealed that the antigen recognized by MAb Ep-C4 was located on cell surface membrane. The membrane antigen of KMS II cells, immunoprecipitated by MAb Ep-C4, was a protein of 81,000 dalton. The reactivity of MAb Ep-C4 was further examined using immunofluorescence and/or immunoperoxidase methods and frozen sections and short-term cultures of various types of brain tumors. No cross-reactivity with normal adult or fetal brain tissues was detected by absorption assay and immunoperoxidase staining. Our results suggest that the antigen defined by MAb Ep-C4 is specific for ependymoma cells, and different from the antigens of glioma cells or other neuroectodermal-derived cells previously described.

[Lateral inferior pontine syndrome: a case report].

The patient, a man aged 57, was admitted to our clinic on May 1, 1987, because of severe vertigo and unsteadiness in standing. Since the age of 55 he had been suffered from hypertension and atrial fibrillation. In September, 1986, he experienced vertigo but recovered soon without therapy. On April 25, 1987, while working, he noticed severe vertigo, nausea and vomiting. He was admitted to a hospital, and then transferred to our clinic. On admission, he was alert and the mentality was normal. Slight ptosis abducent nerve paresis, hypalgesia on the forehead, nose and cheek, facial paresis of peripheral type and hypacusis were detected on the left side. No anisocoria was observed. Sweating was impaired over the left side of the face. Elevation of the soft palate was limited on the left side and the tongue deviated to the left on protrusion. Dysarthria was detected. Though there was no muscular weakness in the extremities, incoordination and dysmetria were noted in the left arm and leg. He could not stand up because of vertigo and unsteadiness. There was no sensory disturbance in the trunk and extremities. Deep tendon reflexes were well elicited and no pathological reflex was observed. These clinical manifestations, except for the ipsilateral palatal and lingual disturbances, were typical of the lateral inferior pontine syndrome caused by occlusion of anterior inferior cerebellar artery, and the lesion was clearly demonstrated by horizontal and coronal MRI. The palatal and lingual disturbances might be due to the involvements of the corticobulbar tracts of the 10th and 12th nerves after the fibers had decussated.

Complete Clq deficiency associated with IgG multiple myeloma.

We report a case of IgG multiple myeloma with selective complete Clq deficiency. The patient was a 75-year-old Japanese woman who exhibited urticaria on the arm and an absence of serum hemolytic complement activity (CH50). Further studies revealed no vasculitis in the urticarial lesion but showed selective complete deficiency of Clq without low molecular weight Clq precipitin. Addition of highly purified Clq restored the CH50 level of the patient's serum to normal. It is suggested that this abnormality was a primary Clq deficiency. We discussed a relationship between the Clq deficiency and myeloma and reviewed the literature.

Myopathy due to glycogen storage disease: pathological and biochemical studies in relation to glycogenosome formation.

Ten cases of myopathy caused by glycogen storage diseases of type II, III, and V, and phosphorylase b kinase deficiency are reported. So-called "abnormal lysosomes" or glycogenosomes which contain abundant glycogen were found in cases of type II, and in some numbers, in cases of type III, and in one case of phosphorylase b kinase deficiency which revealed a moderate decrease in debranching enzyme (amylo-1,6-glucosidase) activity. In these cases of type III and phosphorylase b kinase deficiency, the glycogenosomes are formed through deposition of abnormal glycogen (limit dextrin structure glycogen).

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.

Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells.

The ultrastructural localization of alkaline phosphatase has been examined in cells of a HeLa subline (TCRC-1) that are monophenotypic for Regan isoenzyme expression. Enzyme activity was demonstrated at the cell surface plasma membrane and in certain lysosomes as revealed by the lead citrate method. The regular direct immunoperoxidase procedure utilizing antibodies in IgG or Fab' form showed the same distribution patterns of alkaline phosphatase. However, when the cell surface antigen was blocked in advance with specific unlabeled antibodies and direct immunocytochemistry performed in the presence of saponin, intracellular alkaline phosphatase antigen was observed in the perinuclear space, endoplasmic reticulum, and Golgi apparatus. The results appeared to be concordant with the current concept that membrane glycoproteins are formed in the endoplasmic reticulum, modified in the Golgi apparatus and then transported to the cell surface. Intracellular alkaline phosphatase was observed predominantly in some cell populations especially mitotic cells, suggesting that the enzyme protein was synthesized in and around the mitotic phase. Accordingly, this technique of differential membrane immunocytochemistry appears to provide an opportunity to follow ectopic gene expression as a function of cell cycle and enzyme induction.

Developmentally heterotopic urogenital tissues in a retroperitoneal lipoma with hematopoiesis.

This report concerns an unusual retroperitoneal lipoma with hematopoiesis in a 49-year-old male. A unique feature is the presence of well differentiated male urogenital tissues, such as the prostatic gland, urethra and urinary bladder in part of the tumor. The process of the differentiation of these tissues is also illustrated. Although this lipoma attained a large size weighing 1,500 g, it is apparently a kind of malformation derived from the sequestered retroperitoneal Wolffian vestige. No similar cases have so far been reported in the world literature.

Intracellular alkaline phosphatase activity in cultured human cancer cells.

The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum. Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.

Application of the enzyme histochemical method for the isolation of HeLa variant inducibly producing the Kasahara isozyme of alkaline phosphatase.

A cloning method utilizing the enzyme histochemical procedure was applied to isolate variant HeLa subclones which produce different alkaline phosphatase isozymes. Phosphate was used to distinguish between Kasahara isozyme and Regan isozyme. The use of filter paper made it possible to determine the localization of the Kasahara isozyme-positive colonies. By this method, two variant clones, HeLa S3-10 KP and HeLa S3-10 KN, were isolated. Kasahara isozyme was induced in the former by increased cell density, but not in the latter.

Ultrastructural, cytochemical, and biochemical characterization of alpha-amylase produced by human gastric cancer cells in vitro.

Ectopic production of salivary-type amylase was demonstrated in a human gastric carcinoma cell line (KMK-2) maintained in vitro for more than 3 years. Electron-dense granules, which appeared as zymogens, were observed in the tumor tissue, in cancer cells in the peritoneal fluid (from which the present cell line had been derived), and in cultured cells in early passages. These granules, however, decreased and gradually disappeared during cultivation. Although such a morphologic alteration was recognized, the property of amylase synthesis has been maintained. The presence of alpha-amylase in the cultured cells was demonstrated cytochemically by the immunoperoxidase method. The enzyme secreted and accumulated in the culture medium was partially purified and characterized. Alpha-amylase of KMK-2 cells closely resembled salivary-type amylase in gel filtration profile and disk gel electrophoresis. Immunologic cross-reaction was observed between these enzymes. Secretion into the medium was constant, and the enzyme concentration in the cytoplasm was relatively high when the cells had reached confluence. Prednisolone increased the amylase production two-fold in the cells.

Extramedullary hematopoiesis presenting as mediastinal tumor.

An autopsy revealed extramedullary hematopoietic foci scattered along the thoracic vertebrae and between the ribs in a patient who died of lung cancer. One of them formed a tumor-like mass adhering to the 10th vertebra, and measured 8X6X2 cm. The cellular components included erythrocytic, granulocytic and megakaryocytic series at various stages of maturation as well as abundant iron-pigmented histiocytes. The patient also suffered from chronic hemolytic anemia and secondary hemochromatosis. The intimate relationship of the intrathoracic tumor-like masses of extramedullary hematopoietic tissue with chronic hemolytic anemia is briefly reviewed.

Alkaline phosphatase from two HeLa S3 subclones: one producing Regan and the other, Kasahara isozyme.

HeLa S3 cells have been found to possess heat-stable alkaline phosphatase. However, the present electrophoretic study indicated that this cell line also contained a heat-labile isozyme as a minor component with the major heat-stable (Regan) isozyme. Histochemical demonstration of these two isozymes was also made through heat treatment and inhibition tests. By the single cell cloning of HeLa S3 cells, two subclones, HeLa S3-5 with heat-labile Kasahara isozyme and HeLa S3-10 with Regan isozyme, were established. All 18 subclones from this HeLa S3-5 possessed the same type of heat-labile alkaline phosphatase. These two isozymes were expressed in two cell lines, respectively, during long term culture. The present study suggests that certain cell populations of HeLa S3 have changed their phenotype to Kasahara isozyme due to alteration of a regulatory gene during long term cell culture.

Characterization of liver-type alkaline phosphatase from human gastric carcinoma cells (KMK-2) in vitro.

Alkaline phosphatase was extracted from human gastric carcinoma cells (KMK-2) under long-term culture, and its biochemical and biological properties were investigated. The enzyme was extremely heat labile and was inhibited significantly by L-homoarginine, but only slightly by L-phenylalanine, so that it was classified as a liver-type alkaline phosphatase. Comparative studies with liver and early placental alkaline phosphatases revealed that the enzymes all showed a similar extent of inhibition by amino acids, heat stability, immunological character, molecular, and other biochemical properties. However, KMK-2 alkaline phosphatase was more similar to early placental enzyme in electrophoretic and gel filtration pattern. This liver-type alkaline phosphatase was found ultrastructurally on microvilli of KMK-2 cells, but not on the lateral structurally on microvilli of KMK-2 cells, but not on the lateral surface with interdigitating folds. Prednisolone markedly decreased the content of the present isozyme. Although the present phenotype was stable during long-term culture in regard to the isozyme properties, the original cancer cells from which the cell line had been derived were L-phenylalanine sensitive and moderately L-homoarginine sensitive. This indicated that phenotypic change occurred on cultivation of cancer cells in vitro.

Deviated formation of intestinal glycocalyx in human stomach cancer cells. Another type of signet ring cell.

The process of glycocalyx formation by the trilaminar membrane was investigated at the subcellular level by use of cultivated cancer cells derived from a human stomach adenocarcinoma. Glycocalyx was apparently synthesized on the characteristic trilaminar membrane of Golgi-derived vesicles which gave rise to cytoplasmic vacuoles which, in turn, fused to form an intracytoplasmic cyst. Characteristic microvilli similar to those of intestinal epithelium extended from the membrane lining the intracytoplasmic cyst. These ultrastructural features agree with earlier histochemical findings in suggesting intestinal metaplasia in the origin of the gastric tumor. The morphologic features of the cancer cells clearly indicated that glycoprotein is first synthesized in the Golgi complex and fully formed mucoprotein then emerges as membrane-bound glycocalyx in the vesicles budding from the Golgi stacks. The glycocalyx layer is an integral part of the external leaflet of the characteristic trilaminar membrane. Abundant deposits of glycocalyx in the intracytoplasmic cyst constituted the ultrastructural basis for a distinctive type of signet ring cell that differed from mucous signet ring cells derived from goblet cells.