The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching

An interstitial collagenase was purified from the explant medium of bovine dental pulp and was shown to degrade collagens I and III but not IV and V. The enzyme halted cleft initiation in the epithelium of 12-day mouse embryonic submandibular glands in vitro, indicating the active involvement of interstitial collagens in the branching morphogenesis. Transmission electron microscopic observation of the intact 12-day gland without any clefts showed the scattered localization of a few collagen fibrils at the epithelial-mesenchymal interface of the bulb and also revealed the presence of numerous microfibrils around the stalk. Collagen bundles were regularly seen close to the wavy basal lamina at the bottom of clefts of the intact 13-day gland and 12-day gland cultured for 17 h under normal conditions. Mesenchymal cells were found in the clefts together with the frequent localization of peripheral nerve fibres and capillary endothelial cells. The collagen bundles were more often observed in the 12-day gland cultured in the presence of bovine dental pulp collagenase inhibitor, which had been shown to enhance cleft formation. In contrast, collagen fibrils were rarely found at the epithelial-mesenchymal interface of the 12-day gland cultured in the presence of Clostridial or bovine dental pulp collagenase. The findings indicated that the formation of interstitial collagen bundles is essential to form clefts in the epithelium both in vivo and in vitro.     

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

Summary Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.     

Alpha-interferon in Kikuchi’s disease

In Japan, histiocytic necrotizing lymphadenitis (Kikuchi’s disease) is a relatively common reactive lesion affecting lymph nodes, but the histogenesis and pathogenesis of the disease have not been clarified. Alpha-interferon has a role in the body’s defense against, viral infections. Using a polyclonal antibody to human alpha-interferon, we found numerous cells, mainly histiocytes, containing alpha-interferon in affected foci in the lymph nodes from 24 patients with Kikuchi’s disease. Tubuloreticular structures, thought by some authors to be associated with the production of interferon, were detected by electron microscopy in histiocytes, activated lymphocytes and vascular endothelial cells in the affected foci. These results suggested that the formation of tubuloreticular structures is a secondary phenomenon following stimulation by alpha-interferon. Further, the activity of 2′–5′ oligoadenylate synthetase, which is induced by alpha-interferon and enhanced during the early or active stage of viral infection, showed increased levels of activity in the active stage of Kikuchi’s disease and decreased to normal levels in the convalescent stage 2 weeks later. These results suggested the possibility of a viral etiology for Kikuchi’s disease.     

Multiple rpoD-related genes of cyanobacteria.

Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp. PCC7120, Synechococcus sp. PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp. PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoD1, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal sigma factors of eubacterial strains. The rpoD1 gene showed the greatest similarity to the sigA gene of Anabaena sp. PCC7120.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice ( Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester-linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate-limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Effect of Xyloglucan Nonasaccharide on Cell Elongation Induced by 2,4-Dichlorophenoxyacetic Acid and Indole-3-acetic Acid

Xyloglucan nonasaccharide (XG9) is recognized as an inhibitorof 2,4-D-induced long-term growth of segments of pea stems.In the presence of 10^–5 M 2,4-D, inhibition by 10^–9M XG9 of elongation of third internode segments of pea seedlingswas detected within 2 h after the start of incubation, in someexperiments. Analysis by double-reciprocal (Lineweaver-Burk)plots of elongation in the presence of various concentrationsof 2,4-D, with or without XG9, gave parallel lines, indicatingthat XG9 inhibited 2,4-D-induced elongation in an uncompetitivemanner. XG9 did not influence the 2,4-D-induced cell wall loosening.Thus, XG9 does not fulfill the proposed definition of an "antiauxin". XG9 at 10^–11 to 10^–6 M did not influence IAA-inducedelongation of segments from pea third internodes, azuki beanepicotyls, cucumber hypocotyls, or oat coleoptiles. Inhibitionof IAA-induced elongation by XG9 was not observed even whenthe segments from pea or azuki bean were abraded. Furthermore,fucosyl-lactose at 10^–11 to 10^–4 M did not affectthe IAA-induced elongation of segments of pea internodes orof azuki bean epicotyls. XG9 may be incapable of inhibitingthe IAA-induced cell elongation (especially in oat) or, alternatively,the endogenous levels of XG9 may be so high that exogenouslyapplied XG9 has no inhibitory effect on IAA-induced elongation. (Received February 28, 1991; Accepted May 25, 1991)     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.