Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)     

Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Enzymatic difference between laticifers and cultured cells of papaya

Cell suspension cultures of Carica papaya were found to produce a papain-like enzyme showing an amidase activity similar to papain. Experiments suggested that the enzyme was an SH-enzyme, but an immunological test indicated the absence of papain in cultured cells. The isoelectric point (4.3) of the enzyme of cultured cells was the same as that of the leaf extract, but was different from that of papain or other amidases in the latex of the fruit.     

Spatial relationship among individuals of the Japanese lacertidTakydromus tachydromoides (Sauria,Lacertidae)

The home range ofTakydromus tachydromoides was studied in a grassland area from April 1977 to November 1978. The mean size of home range did not differ markedly between sexes; 136.5 m² for males and 130.8 m² for females. Home ranges of adults overlapped greatly in each sex, and the lizard was considered to be non-territorial. Individuals showed return movement to a definite area (sleeping site) within the home range, and the home range did not shift within a year or between years. Characteristics of the home range of this grassland-inhabiting lizard were discussed in relation to resource abundance and predation pressure.     

Effect of dietary fiber, glucomannan, on absorption of sulfonylurea in man

In order to clarify whether a dietary fiber has any effect upon the intestinal absorption of sulfonylurea, changes in plasma concentration of glibenclamide were determined during a six-hour period in nine healthy volunteers who took 2.5 mg of glibenclamide together with a breakfast and 3.9 g of glucomannan in a form of konjac powder and were compared with those of the control experiment in which the same amount of the hypoglycemic agent was given without the dietary fiber. In the control, mean plasma glibenclamide level increased rapidly, reaching a peak at 60 min and decreased gradually thereafter, whereas an increase in plasma glibenclamide level was blunted in the test experiment, thus plasma concentration of glibenclamide being lower at 30, 60, 90 and 150 min compared with the corresponding value of the control (31.7 +/- 24.5 ng/ml vs 76.4 +/- 25.0 ng/ml at 30 min; 51.3 +/- 35.5 ng/ml vs 120.9 +/- 56.0 ng/ml at 60 min; 60.0 +/- 38.8 ng/ml vs 117.4 +/- 53.1 ng/ml at 90 min; 54.0 +/- 31.5 ng/ml vs 100.7 +/- 46.5 ng/ml at 150 min). Mean plasma glucose concentration was significantly lower at 30 min in the test experiment than in the control despite the lower level of plasma glibenclamide in the former. The results suggest that glucomannan may influence the intestinal absorption of glibenclamide. A dietary fiber must be prescribed in due consideration of these facts.     

Interaction of glycylglycine and Na+ at the mucosal border of Guinea-pig small intestine: A non-mutual stimulation of transport

Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na⁺ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na⁺, while Na⁺ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PD_t), short-circuit current (I_sc) and Na⁺ influx. The change in PD_t (ΔPD_t) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between I_sc and Na⁺ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na⁺, and that translocation of Na⁺ occurs when the two Na⁺ sites on the carrier loaded with Gly-Gly are occupied by Na⁺. However, our results indicate that the resultant Na⁺ flow is not capable of stimulating Gly-Gly translocation.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Energy-requiring uptake of protoberberine alkaloids by cultured cells ofThalictrum flavum

Cultured cells ofThalictrum flavum take up berberine exogenously added to medium against the concentration gradient. This uptake was temperature-dependent and sensitive to plasma membrane-bound ATPase inhibitors such as sodium orthovanadate and diethylstilbestrol, indicating that the process is mediated by an energy-requiring system. The time-course of pH-shift during berberine uptake suggests the participation of a berberine-proton antiport system in the berberine uptake by the cultured cells. In addition, the existence of a specific transport system was suggested by the competitive inhibition of berberine uptake by berberine analogues, coptisine and jatrorrhizine.     

Effect of oxygen supply on berberine production in cell suspension cultures and immobilized cells ofThalictrum minus

The ample supply of O₂ proved to be of great importance for berberine production in cell suspension culture ofThalictrum minus, as the specific O₂ consumption rate of berberine-producing cells was twice as high as that of non-producing cells. Furthermore, berberine yield increased with increases in the volumetric O₂ transfer coefficient (K_La). Estimation of the optimum conditions of oxygen supply in suspension cultures and immobilized cells according to a known theoretical model assuming O₂ uptake by cells to be a zero-order reaction was in good agreement with the experimental data. The O₂ supply to immobilized cells could be improved by reducing the cell density and radius of the bead.     

Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.