Fine structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (cestoda)

Freeze-etch replicas of the protoscolex tegument of Echinococcus multilocularis were examined and compared with conventional thin sections by TEM. The microtopography of the protoscolex tegument was also examined by SEM. The protoscolex consisted of morphologically-distinct, apical and basal tegumentary regions, the latter of which lacked microtriches. The hook area of the apical region contained long, slender, filamentous microtriches that obscured the hook arrangement. These microtriches were structurally different from those found on the suckers and rostellum of the protoscolex. Freeze-etch replicas of the tegumental membrane of the sucker and rostellar microtriches showed that the protoplasmic (P) and exoplasmic (E) faces of the microthrix base and tip contained numerous intramembranous particles (IMP). The densities of the IMP on both the P and E faces of the microthrix tip were approximately twice the number of the larger diameter IMP found on the P and E faces of the microthrix base. No freeze-etch replicas of the microtriches from the hook area were obtained. The basal tegumentary region of the protoscolex consisted of irregularly-distributed, knoblike processes that were variable in size and shape, and contained an electron-dense cap. The IMP on the P face of the knoblike processes measured approximately the same diameter as those on the P face of the microthrix base. However, their density was about half that of the latter. The density of IMP on the E face of the knoblike processes could not be determined from the freeze-etch replicas.     

Ultrastructural immunocytochemical localization of two hydatid fluid antigens (antigen 5 and antigen B) in the brood capsules and protoscoleces of ovine and equine Echinococcus granulosus and E. multilocularis.

The unlabelled antibody method was used in the ultrastructural localization of two hydatid fluid antigens, antigen 5 and antigen B, in brood capsules and protoscoleces of Echinococcus granulosus and E. multilocularis. Antigen 5 was found in the parenchyma cells of the protoscolex and brood capsule wall and to a lesser extent in the walls of the flame cells and collecting ducts of the excretory system and in the surrounding interstitial material. It is suggested that, while some excretion of this antigen may occur from the protoscolex, it could also be liberated into the cystic cavity by degeneration of protoscoleces and parenchymal cells of the brood capsule wall. Antigen B was found mainly in the distal cytoplasm and perinuclear cytoplasm of the tegument anterior to the suckers. It is apparently secreted to the outside and was present in the brood capsule contents; it adheres to the anterior surface and the posterior periodic acid-Schiff (PAS)-positive glycocalyx of the protoscolex and to the inner surface of the brood capsule wall. The protoscolex tegument posterior to the suckers was negative. The parenchyma cells of the protoscolex and brood capsule wall were also positive although the intensity of the reaction product was variable.     

Transfer of proteins synthesized in the cytoplasm into mitochondria. Stimulation of mitochondrial protein synthesis by microsomal fraction]

The in vitro transport into mitochondria of proteins synthesized in the cytoplasm was studied. The system, in which the microsomes synthesize protein in the presence of mitochondria directly during the experiment proved to be the most efficient one. The microsomal fraction significantly stimulated the incorporation of 14C-valine into the isolated mitochondria proteins. The effects of EDTA treatment of the mitochondrial fraction, the dependence of protein synthesis stimulation on the ratio of mitochondria and microsomal proteins and the kinetic pattern of the reaction suggest that the stimulation of the labelled precursor incorporation into mitochondrial proteins is not probably due to the labelled microsomes adsorption on the mitochondria.     

Moniezia expansa: the interproglottidal glands and their secretions

Acetylcholinesterase (EC 3:1:1:7) and alkaline phosphatase (EC 3:1:3:1) were detected in secretions of Moniezia expansa maintained in vitro. Ultrastructural cytochemical studies demonstrated acetylcholinesterase activity on the surface of the microtriches at the base of the interproglittidal glands and in the gland lumen but not in the distal tegument or the gland cells. Alkaline phosphatase activity was demonstrated in the cytoplasm of the gland cells and especially in their protoplasmic connections with the distal tegument. Activity was also found in the distal tegument and the microtriches. It is suggested that the acetylcholinesterase secreted by M. expansa performs a metabolic role at the worm's surface.     

Nitrosylation of cytochrome c during apoptosis

Cytochrome c released from mitochondria into the cytoplasm plays a critical role in many forms of apoptosis by stimulating apoptosome formation and subsequent caspase activation. However, the mechanisms regulating cytochrome c apoptotic activity are not understood. Here we demonstrate that cytochrome c is nitrosylated on its heme iron during apoptosis. Nitrosylated cytochrome c is found predominantly in the cytoplasm in control cells. In contrast, when cytochrome c release from mitochondria is inhibited by overexpression of the anti-apoptotic proteins B cell lymphoma/leukemia (Bcl)-2 or Bcl-X(L), nitrosylated cytochrome c is found in the mitochondria. These data suggest that during apoptosis, cytochrome c is nitrosylated in mitochondria and then rapidly released into the cytoplasm in the absence of Bcl-2 or Bcl-X(L) overexpression. In vitro nitrosylation of cytochrome c increases caspase-3 activation in cell lysates. Moreover, the inhibition of intracellular cytochrome c nitrosylation is associated with a decrease in apoptosis, suggesting that cytochrome c nitrosylation is a proapoptotic modification. We conclude that nitrosylation of the heme iron of cytochrome c may be a novel mechanism of apoptosis regulation.     

An electron microscope study of the tegument of Hunterella nodulosa Mackiewicz and McCrae, 1962 (cestoidea: Caryophyllidea)

Ultrastructural observations using transmission and scanning electron microscopy reveal the tegument to be basically similar to that of other cestodes. The syncytial distal cytoplasm is devoid of organelles except for rod-shaped bodies, believed to be secretory vesicles, and lamellated bodies which probably contribute the raw material for new microtriches. There is evidence that these vesicles originate from the Golgi found in the sub-cuticular cells.Three types of microtriches are described: typical ones with well-developed spines, ones with short filaments instead of spines, and ones with no spines. Microtriches with spines are found only on the anterior part of the worm and may serve to anchor the worm. Microtriches on the posterior have no spines and are believed to be primarily involved in the absorption of nutrients. Between these two regions there is a transitional zone where all three types of microtriches can be found. In general the microtriches are quite uniformly distributed throughout the surface of the worm. The presence of cestodarian-like microtriches raises interesting evolutionary questions.Histochemical tests localized acid and alkaline phosphatase activity on various parts of the tegument, as well as on host intestine, while acian blue tests showed that acid mucopolysaccharide levels correspond with the concentration of the tegument vesicles.     

Echinococcus granulosus: ultrastructure of epithelial changes during the first 8 days of metacestode development in vitro

The epithelium of artificially hatched and activated oncospheres of E. granulosus was studied ultrastructurally over the first 8 days of metacestode development in vitro. Within 4 h of activation, the epithelium was transformed from a thin cytoplasmic layer into a much wider layer packed with penetration gland granules and containing mitochondria and Golgi apparatus. Microvilli were extended from the outer plasma membrane and the basal lamina on the inner epithelial surface virtually disappeared. Microvilli increased in number and length over the first 24 h of development while granules in both the epithelium and penetration gland decreased in number. The granules appear to be involved in microvilli formation. After 3 days of development, the first lamination resolved ultrastructurally as shortened microvilli and some microtriches extending from the epithelium surrounded by an electron-dense microfibrillate material containing sloughed microvilli. By 6 days post-activation, no microvilli remained and only double-walled truncated microtriches extended from the epithelium. The microfibrillate material had become more electron-dense and was closer to the epithelium than at day 1. Within 8 days of metacestode development, a second lamination had developed. Both microfibrillate and particulate material of a greater electron density than the first lamination was added to the microthrix side of the first lamination.     

Taenia taeniaeformis (Cestoda) in the rat: ultrastructure of the host-parasite interface on days 8 to 22 postinfection

The host-parasite interface was examined at the ultrastructural level 8 to 22 days postinfection (DPI) with metacestodes of Taenia taeniaeformis in the rat. Throughout this phase of development the parasite surface was invested with a dense surface coat of complex microtriches. At 8 to 14 DPI the plasma membrane of each microthrix extended beyond the distal end of the electron-dense tip, forming a slender tubular streamer over 10 microns long; by 18 DPI these had shortened and withered. Host cell processes interdigitated with the microtriches without evidence of harm to the parasite surface or the underlying tegument. The cells, on the other hand, became damaged, and their contents were shed into the matrix surrounding the microtriches. Lipid inclusions appeared within the parasites, and in the cytoplasm of surrounding inflammatory cells. By 22 DPI fibroblastic activity had resulted occasionally in the formation of a capsule surrounding a free-floating cysticercus, while in others intense granulocytic infiltration persisted with abutment and intermeshing of host cell and parasite surface processes; however there was still no evidence of any adverse effect on the microtriches, though many granulocytes were clearly pyknotic and degenerating. Evidently, the vigorous cellular response of the host is ineffective in either containing the expansion of the parasite or compromising the integrity of its surface membrane. The changing characteristics of the microtriches may be related to the need for dissolution of both intercellular matrices, and host cells as the vesicular organism rapidly increases in volume.     

Light microscopy and scanning electron microscopy of the In vitro evagination process of Echinococcus multilocularis protoscolices

Marchiondo A. A. and Andersen F. L. 1984. Light microscopy and scanning electron microscopy of the in vitro evagination process of Echinococcus multilocularis protoscolices. International Journal for Parasitotogy14:151–157. During histogenesis of the protoscolices of Echinococcus multilocularis, the apical portion of the protoscolex consisting of the suckers, rostellum and hook region develops as an introversion and invagination within the tissue of the basal portion. In vitro incubation of protoscolices in evagination fluid stimulates the emergence of the apical portion. The initiation of evagination is first detected by a surface change in the basal portion. The smooth contour of this surface which lacks microtriches becomes transformed into tegumental indentations that form transverse and longitudinal furrows within the basal tegument as the protoscolices contract and expand, respectively. An orifice formed at the site or junction where the apical portion is invaginated begins to expand laterally in order to allow emergence of the suckers. The hooks are arranged within the invaginated protoscolex with blades directed towards the basal orifice, the handles directed towards the peduncle and the guards directed laterally. This arrangement persists throughout the evagination of the suckers and rostellum until the apical dome of the hook region emerges, thereby rotating the blades laterally in the direction of the peduncle and rotating the handles and guards medially to assume a coronal arrangement. Evagination is an asynchronous event and therefore allows observation of individual protoscolices in various stages of emergence.     

Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration.

There are few reports concerning the sorting mechanisms of mammalian HSP60 into the mitochondria from the cytoplasm. In the present study we investigated the protein import system. Based on immunoblotting and immuno-histochemistry, HSP60 was detected in both the cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay. HSP60 mRNA was increased in the kidney papilla of rats that had been water restricted for three and five days, but no changes in HSP60 mRNA were detected in the cortex or the medulla of the rat kidneys. Upon immunoblotting, HSP60 was detected in both the cytoplasm and the mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the kidney papilla of rats that were water restricted but the protein was increased in the mitochondria of the rat kidney papilla. We also analysed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with a molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from the affinity column by an excess of signal peptide or by 5 mm ATP. Upon immunoblotting, the 70-kDa protein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytoplasmic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP70.     

Tegument and apical end organ fine structure in the metacestode and adult Proteocephalus ambloplitis

The tegument of plerocercoid and adult P. ambloplitis was examined. Differences in tegument structure existed between these two stages. Plerocercoids of P. ambloplitis lacked extensive vacuolization and unicellular gland cells characteristic of adult tegument. Plerocercoid microtriches were short and conoid; adult microtriches were lenticular with an extended, whip-like shaft. An inclusion, not previously reported from proteocephalid cestodes, is described. Adult tegument had ducts, originating from underlying unicellular glands, extending through the distal cytoplasm and opening to the exterior between microtriches.

The apical end organ cavity of P. ambloplitis contained numerous labyrinth-like spherical bodies. These structures appeared to be synthesized and secreted into the end organ by a thin cellular lining of the end organ. This lining was composed of discrete, filamentous cells believed to be modified subtegumental cell bodies. Spherical structures identical to those observed within the end organ cavity occurred within this cellular lining. The spherical bodies may be associated with enzymes necessary for tissue migration by the metacestode.     

Mitochondrially-imported cytoplasmic tRNA(Lys)(CUU) of Saccharomyces cerevisiae: in vivo and in vitro targetting systems.

The cytoplasmic tRNA(Lys)(CUU) (tRNA(1Lys)) is the single yeast tRNA species to be traffiked from the cytoplasm into the mitochondrial compartment of the cell. To study mechanisms of this targetting we worked out two test systems. The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm. All tRNA species tested were effectively introduced into the cytoplasm, but only the cytoplasmic tRNA(1Lys) was found in the mitochondrial compartment within 1-2 hours after the electroporation procedure. The in vitro system permits specific transfer of the tRNA(1Lys) into isolated mitochondria. Contrary to the known systems for protein transport into isolated mitochondria, mitochondrial import of tRNA(1Lys) in vitro requires the presence of soluble cellular proteins in the reaction mixture. The translocation proved to be ATP-dependent and to require the presence of an ATP-generation system in the reaction. Preincubation of the tRNA with the total cellular extract of the cell markedly increases the rate of the translocation. Two protein fractions are necessary to direct the import in vitro. The first one has high heparin-binding affinity, while the other protein fraction is not retained by heparin-Sepharose.     

Glucose repression and autolysis ofSaccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase

Summary Allerations in the localization of acid phosphatase inSaccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondirial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm.No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.     

Changes in the distribution of mitochondria in mouse embryos blocked at the two-cell stage

Changes in the distribution of mitochondria in the two-cell mouse embryos preceding the developmental arrest in vitro, caused by a genetically determined "two-cell block in vitro" or genisteine treatment, were examined vitally using the mitochondrial-specific probe rhodamine 123 and conventional fluorescence microscopy. In the former case, serious disturbances in the localization of mitochondria appeared already from the middle of two-cell stage, long before the time corresponding to the 2nd cleavage division. Comparison of the behavior of mitochondria in the embryos successfully developing between the one- and two-cell stages and that in the embryos that ceased to cleave suggests that the developmental arrest was accompanied by aggregation of the mitochondria into clusters. There are many such clusters unlike in the cytoplasm of normally developing embryos. Intracellular localization of clusters observed in the genisteine-treated embryos differed radically from that observed in the embryos blocked in vitro at the two-cell stage.     

鱊头槽绦虫原尾蚴皮层的超微结构观察

利用透射电镜观察了头槽绦虫 (Bothriocephalusacheilognathi)原尾蚴皮层的超微结构。头槽绦虫原尾蚴的皮层为典型的合胞体结构。皮层表面有浓密的微毛与少量的突起。与成虫相比 ,原尾蚴的微毛长而且粗 ,呈现单态性 ,推测在进入终末宿主的过程中有脱落和再生现象。突起分布在头部与体侧 ,含有较少的电子致密颗粒。在原尾蚴皮层细胞质中观察到三种腺细胞的分泌过程 ,即顶分泌、外分泌和微分泌过程。原尾蚴身体后部观察到尾钩和穿刺腺管道的开口。在核周区和纤维层下可见发育较好的环肌与纵肌。本文还对原尾蚴皮层突起的功能进行了探讨。     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.     

Mitochondria structural reorganization during mouse embryonic stem cell derivation

Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23–36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~?19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM.     

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.     

Localization of mitochondrial large ribosomal RNA in the myoplasm of the early ascidian embryo

Mitochondrial large ribosomal RNA (mtlrRNA) is transferred out of mitochondria and associates with germinal granules in Drosophila and Xenopus embryos. It has been revealed that mtlrRNA outside of mitochondria is required for formation of the germ-line progenitor, or pole cells in Drosophila. In the present study, the distribution of mtlrRNA was examined in embryos of the ascidian, Halocynthia roretzi, during cleavage stages by whole-mount in situ hybridization. Until the 4-cell stage, the distribution of mtlrRNA coincided with that of mitochondria. which are localized to the cortical cytoplasm in the posterior region of the embryos. Both mitochondria and mtlrRNA were preferentially partitioned into muscle-lineage blastomeres during cleavage stages. After the 8-cell stage, a discrepancy in intracellular localization of mitochondria and mtlrRNA became evident. Mitochondria translocated into central yolkless cytoplasm, while mtlrRNA remained in the posterior cortex in the posterior muscle-lineage b astomeres. The significance of the cortical localization of mtlrRNA in muscle precursor cells in ascidian embryos is obscure. However, the results suggest that mtlrRNA is also transferred out of mitochondria in early ascidian embryos and may play some roles in developmental processes.