Biological effects of extracts obtained from Stryphnodendron adstringens on Herpetomonas samuelpessoai

We report the effect of Stryphnodendron adstringens on the trypanosomatid Herpetomonas samuelpessoai. The parasites were grown at 28 degrees C in a chemically defined medium containing crude extract and fractions at concentrations from 100 to 5000 microg/ml obtained from S. adstringens. Concentrations of 500, 1000, 2500, and 5000 microg/ml both crude extract and semi-purified fraction progressively inhibited the protozoans' growth. At a concentration of 100 microg/ml, crude extract or a semi-purified (F3) fraction did not affect the growth of the protozoans. The F3-9 - F3-12 sub-fractions, at a concentration of 1000 microg/ml, also showed increased inhibitory activity on H. samuelpessoai. The IC50 of the crude extract and the F3 fraction were 538 and 634 microg/ml, respectively. Ultrastructural and enzymatic alterations in the trypanosomatids were also evaluated. H. samuelpessoai cultivated in the presence of IC50 crude extract showed considerable ultrastructural alterations, such as marked mitochondrial swelling with a large number of cristae and evident Golgi complex vesiculation, as observed by transmission electron microscopy. Cells exposed to 538 microg/ml of crude extract at 28 degrees C for 72 h, showed decreased activity of the enzyme succinate cytochrome c reductase, a typical mitochondrion marker, as compared to untreated cells.     

Giant mitochondria distinct from enlarged mitochondria in secretory and ciliated cells of gerbil trachea and bronchioles

Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.     

大鼠心肌线粒体缺血再灌损伤及SOD抗缺血再灌损伤作用的形态计量研究

研究了在体大鼠心肌缺血再灌模型心肌线粒体再灌损伤和SOD抗缺血再灌损伤的作用。实验分为:A组(缺血再灌组)B组(SOD组)和C组(假手术组)。电镜见A组线粒体高度肿胀、外膜缺损、嵴断裂溶解,出现无定形致密体、四膜嵴和杆状嵴等改变。B组线粒体轻度或中度肿胀、少数有轻度嵴溶解。个别线粒体内出现无定形致密体。三组线粒体超微结构立体计量数据比较,A组线粒体密度、线粒体体密度与肌原纤维体密度比率增高((P<0.01),线粒体比表面和比膜面降低(P<0.01)。自身动图像分析仪检测SDH反应灰度值A组呈强损伤反应,B组反应较轻,二者差别有显著性(P<0.05)。结果表明,心肌缺血再灌可致线粒体发生不可逆性损伤,SOD能减轻线粒体缺血再灌性损伤。     

Isoform of Vascular Endothelial Cell Growth Inhibitor （VEGI72-251） Increases Interleukin-2 Production by Activation of T Lymphocytes

The objective of this study is to investigate the characteristics of the recombinant variant ofhuman vascular endothelial cell growth inhibitor,VEGI_(72-251),and compare its biological activities with that ofits prototype VEGI_(24-174),The recombinant plasmid containing the variant VEGI_(72-251) gene was constructedand expressed in Escherichia coli.The effects of the expressed VEGI_(72-251) on cell proliferations were checkedin the human umbilical vein endothelial cell line and certain tumor cell lines (ECV304 and B 16).The inhibitionof VEGI_(72-251) on angiogenesis was detected in the chorioallantoic membrane of chick embryos.In comparisonwith VEGI_(24-174),the recombinant human VEGI_(72-251) seems to have no effect on the proliferation of endothelialcells and the angiogenesis of the chorioallantoic membrane in vitro.An enzyme-linked immunosorbent assay-based method was used for the measurement of interleukin-2 (IL-2) production by peripheral blood monocytes(PBMCs) treated with VEGI_(72-251).PBMCs were pretreated with VEGI_(72-251) (1.25-12.50μg/ml) for 24 h invitro,and the IL-2 concentration in PBMC medium was increased from 354 pg/ml to 1256 pg/ml.It can beconcluded that VEGI_(72-251) is able to increase the level of human IL-2 production by the activation of Tlymphocytes.Differing from VEGI_(24-174) on anti-angiogenesis,VEGI_(72-251) may serve as an anti-cancer factorthrough its activation of T lymphocytes.     

日本七鳃鳗类淋巴细胞的分离及细胞学特征

为分离纯化并鉴定日本七鳃鳗(Lampetra japonica)血液中的类淋巴细胞,采用Ficoll密度梯度离心法分离出单个核细胞层,并得到分离单个核细胞层的最佳分离液比重为1.092。利用流式细胞仪对分离到的单个核细胞层细胞进行分选,根据细胞的前向光及侧向光散射特征成功分选出类淋巴细胞,分选效率为95.68%,每毫升外周血可分离纯化得到类淋巴细胞2.4×106个。通过透射电镜观察七鳃鳗类淋巴细胞,细胞为圆形或椭圆形,细胞表面有突起、无微绒毛,胞质内含有板状嵴线粒体、粗面内质网、游离核糖体和液泡等。淋巴细胞异质性实验结果表明,日本七鳃鳗血液白细胞中尚未发现T淋巴细胞、B淋巴细胞的分化。     

Hepatocellular toxicity of kava leaf and root extracts.

Kava extracts are used widely for different purposes and were thought to be safe. Recently, several cases of hepatotoxicity have been published. To explore possible mechanisms of kava hepatotoxicity, we prepared and analyzed three different kava extracts (a methanolic and an acetonic root and a methanolic leaf extract), and investigated their toxicity on HepG2 cells and isolated rat liver mitochondria. All three extracts showed cytotoxicity starting at a concentration of 50 microg/ml (lactate dehydrogenase leakage) or 1 microg/ml (MTT test). The mitochondrial membrane potential was decreased (root extracts starting at 50 microg/ml) and the respiratory chain inhibited and uncoupled (root extracts) or only uncoupled (leaf extract) at 150 microg/ml, and mitochondrial beta-oxidation was inhibited by all extracts starting at 100 microg/ml. The ratio oxidized to reduced glutathione was increased in HepG2 cells, whereas the cellular ATP content was maintained. Induction of apoptosis was demonstrated by all extracts at a concentration of 150 microg/ml. These results indicate that the kava extracts are toxic to mitochondria, leading to inhibition of the respiratory chain, increased ROS production, a decrease in the mitochondrial membrane potential and eventually to apoptosis of exposed cells. In predisposed patients, mitochondrial toxicity of kava extract may explain hepatic adverse reactions of this drug.     

Towards improved boron neutron capture therapy agents: evaluation of in vitro cellular uptake of a glutamine-functionalized carborane

Abstract  Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. One drawback is the large uptake by the liver causing a decrease of its availability at the tumor region as well as bringing about toxicity problems. A novel carborane-based compound containing a boron payload very similar to that of BSH has been synthesized and tested on rat glioma (C6) cells, hepatoma tissue culture (HTC) cells, and hepatocytes. The newly synthesized system consists of an o-carborane unit (C₂B₁₀H₁₁, o-CB) conjugated to a glutamine residue through a proper spacer, namely, o-CB-Gln. As compared with BSH, it showed the same uptake by C6 cells, but a 50% decrease in uptake by HTC cells and an 80% decrease in uptake by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to that of BSH accompanied by a much lower liver toxicity. Graphical Abstract  A novel carborane based compound, consisting in an o-carborane unit (C2B10H11, o-CB) conjugated to a glutamine residue through a proper spacer (namely o-CB-Gln) has been synthesized, characterized and tested on rat glioma (C6), hepatoma (HTC) and hepatocytes. As compared to sodium borocaptate (BSH), widely used for boron neutron capture therapy (BNCT) of brain tumors, the newly synthesized system showed the same uptake by C6 cells, but a 50% decrease by HTC and 80% decrease by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to BSH accompanied by a much lower liver toxicity.      

5,8,11,14-Eicosatetraynoic acid-induced destruction of mitochondria in human prostate cells (PC-3)

Summary Culturing human prostate PC-3 cells for 4, 24, or 72 h in the presence of 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of arachidonic acid metabolism and cholesterol biosynthesis, markedly altered the morphology and reduced the number of mitochondria in the treated cells. Using quantitative electron microscopic morphometry, we documented changes in the number, form, area, matrix density, and integrity of the cristae and limiting membranes of mitochondria in cells cultured with ETYA. The inhibition of cholesterol synthesis or the substitution of ETYA for polyunsaturated fatty acids in the inner membrane may participate in the disruption of the mitochondria, which resembles the morphologic sequelae of oxidative stress. If sufficiently extensive, these changes could contribute to the inhibition of cellular proliferation by ETYA.     

ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.     

Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis

The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.     

Ultrastructural changes in the spore and mycelia of Ascosphaera apis after treatment with benomyl (Benlate 50 W)

In Ascosphaera apis, after 8 days growth in darkness at 28° C, numerous sporocysts were observed, within which mature spores were seen aggregated into a spore ball. The mature spore of A. apis had a thick spore wall with an electron-opaque outer layer, a spore membrane with many depressions, and sporoplasm containing numerous ribosomes and mitochondria. In the cytoplasm of the mycelium, mitochondria with well-defined cristae and numerous ribosomes were observed. At a concentration of 1 g/ml of culture medium, benomyl appeared to inhibit colony growth of A. apis, but some sporocysts containing deformed spores were found. Deformed spores possessed a thick spore wall with a grainy matrix, and depressions were no longer detected in the spore membrane. Ribosomes were lacking in the sporoplasm and mitochondria appeared degenerate. The mycelium from the treated culture contained mitochondria with an electron-lucid matrix and no well defined cristae, while ribosomes were completely depleted. The significance of these observations in relation to the use of benomyl to control chalkbrood disease in the honey bee is discussed.     

Effects of fetal decapitation on the structure and function of Leydig cells in rhesus monkeys (Macaca mulatta).

Fetal decapitation in utero has enabled us to study the role of fetal pituitary hormones in the development of the fetal testis. Testes from males decapitated near 80 days of gestational life and later delivered at 150 days were smaller than normal and about one-tenth the normal weight. The size of the seminiferous tubules was similar in both groups; however, the number of Leydig cells seemed reduced. In addition, the Leydig cells of the experimental group contained smaller mitochondria with reduced tubular cristae, fewer lipid droplets, and reduced agranular endoplasmic reticulum. Androgen production was inhibited. Measured by radioimmunoassay, the testosterone level in the umbilical vein was 329 +/- 82 pg/ml in six decapitates fetuses, 412 +/- 62 pg/ml in ten normal fetuses. The level in the umbilical artery was 328 +/- 56 pg/ml in five decapitated fetuses, 658 +/- 140 pg/ml in normal fetuses. These studies suggest that chronic deprivation of fetal pituitary hormones inhibits the growth and development of the testis in general and of the Leydig cells in particular.     

Protonophores as inducers of energy-dependent changes of ultrastructure of mitochondria in wheat root cells

The membrane potential of plasmalemma, the release of K⁺ ions into incubation medium, respiratory gas exchange, the ATP content, and changes in the ultrastructure in cells of excised roots of wheat seedlings have been studied under the effect of protonophores 2,4-DNP (2,4-dinitrophenol) and CCCP (carbonyl cyanide m-chlorophenylhydrazone). After 1–4 h, a drop occurred in the plasmalemma membrane potential, as well as the release of K⁺ ions into incubation solution and the suppression of the intensity of oxygen absorption by cells. Mitochondria were of ovoid shape and had numerous and clearly outlined, slightly swollen cristae, which corresponds to the condensed type of the organelles. Additionally, a peculiar spatial arrangement of cristae in mitochondria has been revealed (as piles parallel to each other, as well as in the form of fans and of propellers) under the effects of protonophores. After 5 h of the action of protonophores against a background of the significant stimulation of oxygen consumption, low membrane potential, and a decrease in the functional activity of mitochondria, the destruction of the cell ultrastructure began. It is suggested that the revealed conformational transitions of mitochondria reflect gradual changes in their functional activity and the functional state of the cells under the long action of protonophores.     

Localization of ryanodine receptor 3 in the sinus endothelial cells of the rat spleen

The ultrastructural localization of ryanodine receptors (RyR) in sinus endothelial cells of the rat spleen was examined by confocal laser scanning and electron microscopy by using isoform-specific antibodies to each of the RyR isoforms. Immunofluorescence microscopy of tissue cryosections revealed RyR3 to be localized, with a strand-like form, in the superficial layer and within the cytoplasm of endothelial cells. Antibodies to RyR1 and RyR2 did not react indicating RyR3 was the predominant isoform. RyR3 was observed over the cortical layer of actin filaments in the apical part and beneath stress fibers in the basal part of the endothelial cells. The distribution of Ca²⁺-storing tubulovesicular-structures within endothelial cells was established by tissue sections treated with osmium ferricyanide selectively to stain the sarcoplasmic reticulum and transverse tubules in muscle cells; electron microscopy revealed densely stained tubulovesicular structures located throughout the sinus endothelial cells and interconnected at various sites. These structures closely apposed the plasma membrane at the apical, lateral, and basal surfaces of the cells and occasionally ran closely parallel to the plasma membrane and near to the mitochondria. Immunogold electron microscopy revealed RyR in the membranes of the nucleus, tubulovesicular structures, and subplasmalemmal cisternae. In the subplasmalemmal cisternae at the apical, lateral, and basal surfaces, RyR was detected on the membranes near to the plasma membrane. Labeling was also present on the membranes of tubulovesicular structures near to caveolae and on the cristae of the mitochondria. Thus, RyR probably participates in Ca²⁺ signal transduction and/or mechanosignal transduction in sinus endothelial cells.This work was supported by Grant-in-Aid for Scientific Research (C), Japan.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Microcalorimetric study of the effect of Ce(III) on metabolic activity of mitochondria isolated from indice rice 9311

Rare earth elements (REEs) have beneficial influence on plant growth and are widely used in agriculture practice, but little is known about behavior of the REEs on mitochondria in plant cells. Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from indice rice 9311 (Oryza sativa L.), and the effect of Ce(III) on mitochondrial metabolism was investigated. By analyzing the obtained heat flux curves, the crucial parameters such as activity recovery rate constant (k) and maximum heat power (P(m)) were investigated. Application of Ce3+ in concentrations ranging from 0 to 120 microg/ml significantly increased k and P(m) values, with the maximum reaching 261 and 180% of the control, respectively. Concentrations from 140 to 150 microg/ml had the opposite effect. These results were consistent with previous reports on the effects of REEs on plant growth. It was concluded that the Ce(III)-induced change of mitochondrial metabolic activity is a possible mechanism by which Ce(III) influenced indice rice 9311 growth.     

Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation

Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria.     

Coupling factor B affects the morphology of mitochondria

Ectopic expression of coupling factor B in animal cells resulted in altered mitochondrial morphology. Cells expressing factor B fused to green fluorescent protein (GFP) contained fragmented, balloon-shaped or thinned, filamentous mitochondria, terminating at one end with balloon-like structures. Ultrastructural analysis using transmission electron microscopy revealed changes in the organization of mitochondrial cristae in cells expressing factor B-GFP fusion protein.     

Effects of PVA and PEG Pretreatment on Development and Ultrastructure of Plumular Root Mitochondria in Soybean Seed During Low Temperature Imbibition Process

Electron microscope observation revealed that mitochondria in plumular root cells of soy bean seed were invisible at the beginning of seed germination. They were sequentially formed re-differentiated and developed from proplastids during the process of germination. The proplastids in the control plumular root markedly expanded at low temperature imbibition process and did not develop into mitochondria when normal temperature was restored. Even the structure of reticulata membrane was further destroyed and vacuolized. In contrast, mitochondria of soybean seed pretreated with polyvinyl alcohol (PVA) and polythylene glycol-6000 (PEG) continused to develop well under the same conditions, as characterized by a clear structure of bilayered membrane and tube cristae. The results suggest that continuous re-differentiation and development of proplastids into mitochondria at low temperature imbibition process play an important role in the increase of soybean seed vigour and its resistance to chilling injury.