Respiration-dependent Calcium Transport in the Cold-shocked Goat Spermatozoa

寒冷衝撃による山羊精子の原形質膜の損傷は,ひいては原形質膜のイオン選択透過性の低下をもたらしているものと考えられる. そこで,本研究は寒冷衝撃不動化山羊精子の原形質膜の損傷を,Ca2+依存性酸素消費の亢進を測ることによって明らかにしようとした. 1.NaF不動化精子と正常精子の酸素消費量は,添加Ca2+によって影響されなかった. また,DNP添加によって脱共役効果が認められた. 2.寒冷衝撃を施した不動化精子の酸素消費量は,添加Ca2+の添加量(1mM以下)に応じて段階的に亢進された. また,その酸素消費量の亢進,すなわち,Ca2+の取り込みはDNPによってわずかに阻害された. 高濃度(1mM以上)の添加Ca2+は阻害的に作用した. 3.寒冷衝撃時に卵黄物質が存在することにより,その酸素消費量はCa2+添加によって影響されなかった. 寒冷衝撃は,山羊精子における添加Ca2+依存性酸素消費量の亢進を示し,このことは原形質膜でのCa2+の選択透過性の低下をもたらしているものと思われた. また,寒冷衝撃時の卵黄物質の存在によって,原形質膜の機能低下が防がれることが推察された

Fluorescence and electron microscopic study of intracellular F-actin in concanavalin A-treated and cytochalasin B-treated Ehrlich ascites tumor cells.

To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones.</p

Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells

Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G.-glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G.-glabra

Inhibition of Phospholipase A2 and Platelet Aggregation by Grycyrrhizin, an Anti-inframmation Drug

We studied the effect of glycyrrhizin, a compound known as an anti-inflammatory and antiallergic drug, on the membrane permeability change induced by phospholipase A2 (PLA2) and on platelet aggregation. Glycyrrhizin was found to inhibit the PLA2-induced carboxyfluorescein (CF) release from D,L-dipalmitoyl phosphatidylcholine (DPPC) liposomes. Part of this inhibitory effect of glycyrrhizin on PLA2 is accounted for by the physical state of the substrate, the DPPC liposome membrane. Glycyrrhizin also inhibited collagen-induced platelet aggregation in a concentration dependent manner, which may in part account for its inhibitory effect on PLA2.</p

Utilization of the ability to induce activation of the nuclear factor (erythroid-derived 2)-like factor 2 (Nrf2) to assess potential cancer chemopreventive activity of liquorice samples

Introduction – Nuclear factor (erythroid-derived 2)-like factor 2 (Nrf2) is a transcription factor that regulates expression of many detoxification enzymes. Nrf2-antioxidant responsive element (Nrf2-ARE) signalling pathway can be a target for cancer chemoprevention. Glycyrrhiza glabra, common name, ‘liquorice’, is used as a sweetening and flavouring agent, and traditionally, to treat various ailments, and implicated to chemoprevention. However, its chemopreventive property has not yet been scientifically substantiated. Objective – To assess the ability of liquorice root samples to induce Nrf2 activation correlating to their potential chemopreventive property. Methods – The ability of nine methanolic extracts of liquorice root samples, collected from various geographical origins, to induce Nrf2 activation was determined by the luciferase reporter assay using the ARE-reporter cell line, AREc32. The antioxidant properties were determined by the 2,2-diphenyl-1-picryhydrazyl (DPPH) and the ferric-reducing antioxidant power (FRAP) assays. Results – All extracts exhibited free-radical-scavenging property (RC50 = 136.39-635.66 g/mL). The reducing capacity of ferrous ion was 214.46-465.59 M Fe(II)/g. Nrf2 activation indicated that all extracts induced expression of ARE-driven luciferase activity with a maximum induction of 2.3 fold relative to control. These activities varied for samples from one geographical location to another. Conclusions – The present findings add to the existing knowledge of cancer chemoprevention by plant-derived extracts or purified phytochemicals, particularly the potential use of liquorice for this purpose

Survival of Frozen Rat Embryos : Ice Formation on Rat Blastocyst during Freezing Process

8% DMSOあるいは5% グリセリン添加のラット胚盤胞の凍結後の生存性を検討した. 8% DMSOを加え,DAからLN2ガス10分静置後LN2に浸漬したものが最も良好な成績が得られ,5% グリセリンを加え,DAから同様の法によるものは著しく劣った. いずれもDAから直接LN2へ浸漬した場合は生存胚は得られなかった. 細胞内凍結を観察するために,凍害防御物質を含まない液でラット1cell胚を凍結し,低温顕微鏡によって観察したが,Flashing直後は不透度が急激に増し,数分後には移動再結晶による不透度の低下を招いた. ラット胚盤胞を同様に凍結した場合,-20℃で細胞外氷晶の移動が止まり,卵細胞の萎縮はわずかで原形に近い形で固定され細胞内凍結が想像された. 5% グリセリン加媒液で凍結した場合,移動再結晶誘起後の緩冷によって細胞外氷晶の増大と胚盤胞の萎縮が観察された. しかし,その氷晶形の移動とその変化は,-80℃以下では認められず,胚は脱水不充分のままでそれ以下の温度で細胞内凍結を招来していることが推察された. 8% DMSO加媒液で凍結した場合,氷晶の形成と移動はグリセリンと同様の経過を示したが,-80℃からDMSOの共晶点まで細胞外氷晶の角化と増大が認められ,胚の萎縮もその温度まで続いた. さらに-150～160℃付近において氷晶形の配列とは無関係に氷晶部に亀裂が入り,それ以後,-197℃まで変化は認められなかった. また,細胞内の不透度の変化は全温度域を通じて認められず,細胞内凍結はごく一部かあるいは生じていないことが想像された

Survival of Frozen Rat Embryos : Ice Formation on Rat Blastocyst during Freezing Process

8% DMSOあるいは5% グリセリン添加のラット胚盤胞の凍結後の生存性を検討した. 8% DMSOを加え,DAからLN2ガス10分静置後LN2に浸漬したものが最も良好な成績が得られ,5% グリセリンを加え,DAから同様の法によるものは著しく劣った. いずれもDAから直接LN2へ浸漬した場合は生存胚は得られなかった. 細胞内凍結を観察するために,凍害防御物質を含まない液でラット1cell胚を凍結し,低温顕微鏡によって観察したが,Flashing直後は不透度が急激に増し,数分後には移動再結晶による不透度の低下を招いた. ラット胚盤胞を同様に凍結した場合,-20℃で細胞外氷晶の移動が止まり,卵細胞の萎縮はわずかで原形に近い形で固定され細胞内凍結が想像された. 5% グリセリン加媒液で凍結した場合,移動再結晶誘起後の緩冷によって細胞外氷晶の増大と胚盤胞の萎縮が観察された. しかし,その氷晶形の移動とその変化は,-80℃以下では認められず,胚は脱水不充分のままでそれ以下の温度で細胞内凍結を招来していることが推察された. 8% DMSO加媒液で凍結した場合,氷晶の形成と移動はグリセリンと同様の経過を示したが,-80℃からDMSOの共晶点まで細胞外氷晶の角化と増大が認められ,胚の萎縮もその温度まで続いた. さらに-150～160℃付近において氷晶形の配列とは無関係に氷晶部に亀裂が入り,それ以後,-197℃まで変化は認められなかった. また,細胞内の不透度の変化は全温度域を通じて認められず,細胞内凍結はごく一部かあるいは生じていないことが想像された