The role of calcium ions in freezing injuries of winter oilseed rape leaves. Effects of calcium channel blockers and mimesis by calcium ionophore

Calcium ionophore A23187 allowing for a calcium ion influx from an apoplast to a cytoplasm, mimicked symptoms of the frost-induced injuries in winter oilseed rape leaves, as estimated by the conductivity method. Both calcium ionophore and freezing treatment induced degradation of phosphatidylcholine. On the other hand lanthanum and gadolinum ions as well as verapamil, the inhibitors of calcium ion channels, decreased the degree of the frost-induced injuries. Lanthanum ions prevented the frost-induced degradation of PC. It is proposed that freezing alters the functioning of calcium ion channels which results in calcium ion influx into a cytosol. This in turn may lead to a degradation of cell membranes.     

草履蚧血细胞的显微形态与超微结构观察

为研究蚧虫血淋巴中血细胞的种类及其结构特征,采用相差显微镜、荧光显微镜和扫描电镜与透射电镜技术,观察了草履蚧雌成虫血细胞的显微形态与超微结构特征.结果显示,在荧光显微镜和扫描电镜下观察识别出草履蚧血淋巴中的5种血细胞,即：原血胞、浆血胞、粒血胞、囊血胞和类绛色血细胞.在透射电镜下可见,原血胞的细胞核明显,表现出高的细胞质密度;浆血胞最典型的特征是细胞质中有大量的囊泡;粒血胞的细胞质中有发达的粗面内质网和许多玫瑰形的细颗粒.类绛色血细胞最典型的特征是有许多结晶,并在细胞质的空泡区内分布的更多.囊血胞透明性强,具有围核空间,膨大成潴泡状.     

Cytophysiological features of hemocytes in cockroaches

Summary The wide range of functional activities of circulating and sessile insect hemocytes expresses itself in highly specialized cytological terms. Electron microscopic studies carried out in five species of normal and experimentally manipulated cockroaches, in conjunction with light microscopic information, reveal a broad spectrum of structural organization and an apparent capacity for cellular modulation. In addition to conventional organelles, these hemocytes contain a class of unusual cytoplasmic inclusion bodies which seem to undergo striking transformations in response to specific functional demands. A variety of transitional forms attests to the existence of close links between tubule containing (type 1), electron dense (type 2), and large globular (type 3) inclusion bodies, and reveals the derivation of yet another special (lamellated, fusiform) inclusion from type 2 bodies. Confluence of the type 3 configurations into still larger lacunae may precede the release of their contents into the hemolymph, a process whose major effect seems to be the initiation of the clotting process.Another important activity of hemocytes concerns the programmed reorganization of the stromal framework of the various organs. The dominant feature of blood cells engaged in the deposition of connective tissue are greatly distended cisternae of the rough endoplasmic reticulum and accumulations of banded fibrils at the interface of cytoplasm and extracellular space. The engulfment of discarded stromal material can be visualized in fortuitous sections representing steps in its incorporation by hemocytes. Ultrastructural correlates of the breakdown of these and other disintegrating or noxious elements by certain hemocytes are prominent digestive vacuoles with heterogeneous contents and reaction products of hydrolytic enzymes. The capacity for the uptake of small particles by micropinocytosis is demonstrated by the localization of horseradish peroxidase activity at the cellular surface and within cytoplasmic vesicles.The diversity of structural appearances reflects a division of labor, while the many transitional features of hemocyte morphology favor the concept of functional flexibility of one basic cell type rather than a strict classification into distinctly separate cellular types.Supported by research grants NB-05219, NB-00840, and 5 P01 NS-07512 from the U.S.P.H.S.I am greatly indebted to Dr. Joseph Osinchak, City University, New York, who has generously permitted me to include some of his micrographs in the present study. I also want to express my gratitude to Mrs. Sarah Wurzelmann and Mr. Stanley Brown for their excellent technical assistance.     

Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

幼虫密度对草地螟血细胞数量和组成的影响

为了阐明幼虫密度对草地螟Loxostege sticticalis L.（鳞翅目： 螟蛾科）细胞免疫能力的影响, 本研究调查了在活体灰菜植株上1,5,10和20头/瓶（900 mL）4种密度条件下的其5龄幼虫血细胞种类、数量和组成。结果表明： 草地螟幼虫血淋巴中有原血细胞、浆血细胞、 颗粒血细胞、珠血细胞和类绛色血细胞等5种（类）血细胞。血细胞总数、 浆血细胞、颗粒血细胞数量随幼虫密度的增加而显著递增, 但原血细胞、珠血细胞和类绛色血细胞数量在幼虫密度间的差异不明显;各种血细胞所占血细胞总数的比例在4个密度中的排序相同, 但10和20头/瓶密度下的浆血细胞比例显著高于1头/瓶的,其余4种血细胞的比例在不同密度之间无显著差异。可见, 幼虫密度主要是通过影响草地螟幼虫浆血细胞和颗粒血细胞的数量及血细胞总数, 从而影响草地螟的细胞免疫能力。     

Effect of the cyclopeptolide 90-215 on the production of destruxins and helvolic acid by Metarhizium anisopliae

The effect of the cyclopeptolide 90-215 on the production of destruxins and helvolic acid in various Metarhizium anisopliae strains was investigated. Addition of 10.0 mg L⁻¹ of the cyclopeptolide to the production media increased the production of destruxins by 1.3-fold to 12.5-fold, whereas the production of helvolic acid was decreased by 1.6- to 11.0-fold. Fifty liters were fermented in Erlenmeyer flasks with the strain 86-23766 grown in medium supplemented with cyclopeptolide 90-215. The procedure for isolation and purification of destruxins was simplified due to the higher yield of destruxins. Good quantities of destruxins were obtained from this fermentation. The results of our studies show that the addition of small quantities of a suitable compound can drastically alter the production and relative ratios of secondary metabolites. This can have a wide range of potential applications in the area of metabolite production. Received 09 December 1996/ Accepted in revised form 09 April 1997     

Effect of Cry1Ab protein on hemocytes of the wolf spider Pardosa pseudoannulata

Studies on the effect of Cry1Ab protein on hemocytes of the wolf spider Pardosa pseudoannulata revealed that Cry1Ab protein could accumulate in the four-instar and adult spiders via the food chain from transgenic rice expressing Cry1Ab protein through its prey brown planthoppers with approximate 20-time enrichment, but could not accumulate in hemolymph of the spider. The accumulated Cry1Ab had no significant effects on several elements of hemolymph including stored energy, calcium ion concentration and apoptosis rate of the hemocytes, indicating that Cry1Ab could not affect the hemocytes of P. pseudoannulata.     

红褐斑腿蝗血细胞的形态与分类

利用光学显微镜和显微数码拍照系统,对红褐斑腿蝗Catantops pinguis(Stal)血细胞的形态进行观察和分类。结果在红褐斑腿蝗血淋巴中观察到5种血细胞,分别是原血胞、浆血胞、粒血胞、珠血胞和囊血胞。原血胞为小型圆形细胞,边缘圆滑、清晰,核质比例很大。粒血胞多为中型,形状不规则,边缘凹突不平,内含较大的异质性溶酶体颗粒。浆血胞多为中型,刚离体时形状较规则,常呈圆形、卵圆形。浆血胞内缺乏大的颗粒,细胞核大而圆形,细胞质内具许多小型颗粒状物质。浆血胞离体后形状变化较多,常发展出伪足,呈丝状、短芒状、钩状或片状伪足。珠血胞多为大、中型,外形大体呈圆形,但边缘由于大小不等的珠形内含物突出,呈花瓣状。囊血胞多为中型,圆形或椭圆形,细胞质内具有大小不一的带有折光性的颗粒或块状物,细胞边缘比较光滑。     

罗氏沼虾与克氏原螯虾血细胞的比较研究

对罗氏沼虾与克氏原螫虾血细胞的分类与组成进行了染色观察比较研究。根据染色后光镜下血细胞的核质比、颗粒的大小和数量等来对血细胞进行分类,罗氏沼虾与克氏原螯虾的血细胞均可分为透明细胞、半颗粒细胞和颗粒细胞三类;其血细胞浓度分别为1.02±0.21×10⁷ Ind·ml^-1和0.85±0.15×10⁷Ind·ml^-1。2种虾血细胞的颗粒形态存在显著差异;透明细胞、半颗粒细胞和颗粒细胞占血细胞总量的百分比在罗氏沼虾为21.3±6.3%,45.7±2.5%,33.0±6.8%:在克氏原螯虾为12.0±5.8%、49.5±5.1%和38.5±9.5%。     

Interference by edta and calcium ions of the 3,5-dinitrosalicylate reducing sugar assay

The ability of EDTA and calcium ions to interfere with the 3,5-dinitrosalicylate reducing sugar assay; is reported. The antagonistic efrects of Ca²⁺ and EDTA are evaluated.     

In vitro effect of fungal cyclodepsipeptides on leukemic cells: study of destruxins A, B and E.

The activities of three mycotoxins isolated from the hyphomycete Metarhizium anisopliae: destruxin A, B, and E (DA, DB and DE) are described and compared in vitro on leukemic cells. Their antitumor effect was investigated by flow cytometry on growth, cell viability and cell cycle perturbation 48 h after destruxin exposure. Against P388 leukemic cells, DE displayed greater antiproliferative activity than DA and DB. The minimum concentration required to inhibit 50% of cell proliferation is 0.33 microgram/ml for DE, 11.7 micrograms/ml for DA and 9.4 micrograms/ml for DB. Cell cycle modifications were only observed with DE at 50 and 10 micrograms/ml and consisted in an accumulation of the cells in G0/1 phase. DA and DB did not modify the number of cells in G0/1 of the cell cycle. Nevertheless a decrease in the number of cells in G2+M phase was induced by the three destruxins.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

Kinases,intracellular calcium,and apolipophorin-III influence the adhesion of larval hemocytes of the lepidopterous insect,Galleria mellonella

Based on the results from the use of selective inhibitors and activators, active protein kinase A, protein tyrosine kinase, and protein kinase C (PKC) isoforms decreased the adhesion of larval Galleria mellonella hemocytes to glass slides. The protein kinase A inhibitor at all concentrations increased granular cell adhesion only whereas protein tyrosine kinase elevated both granular and plasmatocyte attachment at the lowest concentration. Active, Ca(2+)- and lipid-dependent PKC isoforms limited plasmatocyte and granular cell adhesion whereas PKC that was inhibited by selected compounds (with differed modes of PKC inhibition) enhanced hemocyte attachment. The granular cells were more sensitive to the PKC inhibitors than were plasmatocytes. Phospholipase C and its diacylglyceride product were necessary to reduce hemocyte adhesion and maintain PKC activity. Extracellular Ca(2+), possibly transported through L-channels, was required for plasmatocyte attachment. In contrast, lowering the levels of cytosolic Ca(2+) was associated with decreased PKC activity and was required for hemocyte adhesion.     

Chemotactic attraction between hemocytes of the oyster,Crassostrea virginica,and bacteria

Experiments were conducted to ascertain whether there is chemotactic attraction by Bacillus megaterium and Micrococcus varians, both Gram-positive species, and Escherichia coli and Vibrio parahaemolyticus, both Gram-negative species, for hemocytes of the American oyster, Crassostrea virginica. It was ascertained quantitatively that oyster hemocytes are attracted to live E. coli, B. megaterium, and M. varians but not to heat-killed bacteria. Furthermore, oyster cells are not attracted to either live or heat-killed V. parahaemolyticus. It is concluded that the chemoattractant is some molecule emitted by living vegetative cells of certain Gram-positive as well as Gramnegative bacteria.     

虾类血细胞的分类与功能研究进展

血细胞在动物的免疫防御体系中扮演了重要的角色,尤其是对缺少适应性免疫的无脊椎动物.在这些动物中,血细胞既参与细胞免疫的吞噬、包囊、结节等作用,还参与体液免疫中许多免疫因子的生成与储存.对不同动物类群的血细胞的不同亚群进行区分,是深入了解其免疫机制与血细胞功能的基础.尽管国内外学者利用不同的方法,针对虾类血细胞的不同亚群...     

Involvement of cAMP-Dependent protein kinase and intracellular free calcium ions in regulation of ovulation of the follicle-enclosed oocytes ofRana temporaria by gonadotropic hormones

The inhibitor of cAMP-dependent protein kinase N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) (12.5–50 μM) decreased the rate of ovulation of the follicle-enclosedRana temporaria oocytes induced by the homologous pituitary extract in amphibian Ringer solution and in a chloride-free medium. The inhibitor of voltage-dependent calcium channels diltiazem (10 and 100 μM) decreased the rate of ovulation in Ringer solution but did not affect it in a chloride-free medium or decreased the ovulation inhibitory effect of this medium. It was concluded that cAMP-dependent protein kinase and intracellular free calcium ions were involved as second messengers in the gonadotropin regulation not only in maturation of amphibian oocytes but also in ovulation.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.