Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

Effects of Physical Parameters upon Protoplast Electrofusion

The effects of three physical parameters upon protoplast electrofusionwere studied using protoplasts from cultured cells of Coptisjaponica and Euphorbia millii. The osmotic potential of themedium did not appreciably affect the AC-field-induced protoplast-pairformation, but significantly influenced the fusion process ofthe paired protoplasts in response to DC pulses. The optimumosmotic potential was 0.55 to 0.60 Osm/kg H₂O in our system.The density of the medium markedly influenced both pair formationand fusion process. The optimum density was 1.13 to 1.14 g/cm₃,and at this density the yield of the fused protoplasts increasedto more than twice that of the control (1.10 g/cm₃). Hydrophiliccoating of the bottom surface of the chamber with Gellan gumor polyacrylamide gel was also effective for both pair formationand the fusion process, while coating with hydrophobic siliconewas entirely inhibitory. Possible interpretations of the effectsof these physical parameters upon protoplast electrofusion arepresented. ¹Permanent address: Biochemical Research Laboratories, KanegafuchiChemical Industry Co., Ltd., Takasago, Hyogo 676, Japan. (Received December 21, 1987; Accepted March 18, 1988)     

An assessment of nasal functions in control of breathing

Breathing pattern and steady-state CO2 ventilatory response during mouth breathing were compared with those during nose breathing in nine healthy adults. In addition, the effect of warming and humidification of the inspired air on the ventilatory response was observed during breathing through a mouthpiece. We found the following. 1) Dead space and airway resistance were significantly greater during nose than during mouth breathing. 2) The slope of CO2 ventilatory responses did not differ appreciably during the two types of breathing, but CO2 occlusion pressure response was significantly enhanced during nose breathing. 3) Inhalation of warm and humid air through a mouthpiece significantly depressed CO2 ventilation and occlusion pressure responses. These results fit our observation that end-tidal PCO2 was significantly higher during nose than during mouth breathing. It is suggested that a loss of nasal functions, such as during nasal obstruction, may result in lowering of CO2, fostering apneic spells during sleep.     

Small cell carcinoma of the stomach: an immunohistochemical and electron microscopic study.

A 4 x 6 cm ulcerative mass in the antrum was found to consist of papillary adenocarcinoma in the surrounding wall and the small round cell neoplasm at its base. Immunohistochemical staining revealed that elements of the papillary adenocarcinoma were positive for carcinoembryonic antigen, epithelial membrane antigen, keratin, endocrine granule constituent, and CA19-9, while components of the small cell carcinoma were weakly positive only for neuron-specific enolase. In one portion of the small cell carcinoma, particularly large cells with pleomorphic nuclei which were intensely positive for desmin were detected. Electron microscopic examination revealed dense-cored granules and intercellular junctions in the small neoplastic cells and bundles of intermediate filaments in the desmin-positive large cells. These findings suggest that ultrastructural examination is vital in diagnosis of small cell carcinoma and they reveal the capability of this carcinoma toward multidirectional differentiation.     

Efficiency of particle-bombardment-mediated transformation is influenced by cell cycle stage in synchronized cultured cells of tobacco

Plasmid DNA pB1221 harboring β-glucuronidase gene was delivered to synchronized cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells of different cell cycle stages by a pneumatic particle gun. The cells bombarded at M and G₂ phases gave 4 to 6 times higher transformation efficiency than those bombarded at the S and G₁ phases.     

Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.     

On the origin of neoblasts in freshwater planarians (Turbellaria)

Experiments on 1) regeneration of the cave-adapted planarian, Sphalloplana zeschi, 2) induction of sexuality in an asexual strain of Dugesia japonica japonica by feeding, and 3) culture of dissociated planarian cells, show that neoblasts originate from intestinal cells, i.e. phagocytic cells and granular clubs.     

Sensitivity of preadipose 3T3 cells to growth hormone

Cultured preadipose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3-F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.     

Studies on the ontogenesis and the regulatory mechanisms of placental lactogen and insulin binding sites (receptor) in rat liver

The ontogenesis of specific binding of 125I-hPL and 125I-insulin was determined in rat liver cell membranes (10(5) X g pellets), and the regulatory mechanisms of these binding sites were also examined. There were striking differences in the mode of ontogenesis between binding sites of hPL and insulin in rats. HPL binding sites were very few in liver cell membranes from fetal and immature rats. They began to increase after puberty, and markedly increased in late pregnancy. On the other hand, insulin binding sites, which decreased in late pregnancy, were dominant in fetal liver and placenta. Consequently, the lipolytic and glycogenolytic activities of hPL in maternal liver were accentuated, whereas the effects of insulin on maternal liver were suppressed. In contrast, in fetal liver and placenta only the anabolic effects of insulin seemed conspicuous. According to the results of experiments on in vivo administration of estradiol-17 beta, progesterone, hydrocortisone or hPL to intact or hypox-rats, and the measurement of serum rat chorionic mammotropin (rCM), rPRL, estradiol-17 beta, and insulin during pregnancy in rats, the increase in hepatic hPL binding sites observed in late pregnancy might be, at least in part, due to rCM secreted from placenta, and the decrease in insulin binding sites due to the increase in serum insulin itself in rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture