Freeze-fracture study of the site of attachment of Cryptosporidium muris in gastric glands.

The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Multilocus Sequence Subtyping and Genetic Structure of Cryptosporidium muris and Cryptosporidium andersoni

In this study, nine C. muris and 43 C. andersoni isolates from various animals in China were subtyped by a multilocus sequence typing (MLST) tool. DNA sequence analyses showed the presence of 1-2 subtypes of C. muris and 2-6 subtypes of C. andersoni at each of the four loci (MS1, MS2, MS3, and MS16), nine of which represented new subtypes. Altogether, two C. muris and 10 C. andersoni MLST subtypes were detected. Linkage disequilibrium analysis indicated although the overall population structure of the two parasites was clonal, the Chinese C. andersoni in cattle has an epidemic structure. Three and two clusters were produced in the C. muris and C. andersoni populations by Structure 2.3.3 analysis, with Chinese C. muris and C. andersoni substructures differing from other countries. Thus, this study suggested the prevalence of C. andersoni in China is not attributed to the introduction of dairy cattle. More studies involving more genetic loci and systematic sampling are needed to better elucidate the population genetic structure of C. muris and C. andersoni in the world and the genetic basis for the difference in host specificity among the two most common gastric parasites.     

Distribution of actin and tropomyosin in Cryptosporidium muris

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (×2,000). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various cellular functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites'' firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.     

Freeze-fracture studies of frog atrial fibres.

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the "gap junction" or "nexus". The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.     

Isolation and identification of Cryptosporidium from various animals in Korea. II. Identification of Cryptosporidium muris from mice

Each of SPF mice(Scl: ICR strain, 3-week-old males) was inoculated with 5 x 10(4) oocysts of Cryptosporidium by stomach tube. The oocysts were large type one which was previously isolated from Korean mice, and passaged in 3-week-old SPF mice. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of developmental stages the stomach of the mice was examined by transmission electron microscopy (TEM) at 4 weeks post-inoculation. The prepatent period for 6 mice was 5.6 days post-inoculation on the average, and the patent period was 63.2 days. The number of oocysts discharged per day from the mice reached peak on day 36.6 post-inoculation on the average. A large number of oocysts were found in fecal samples obtained from inoculated mice on days 30-50 post-inoculation. C. muris was larger than C. parvum at almost every developmental stages, the size difference being 1.4 times in oocysts, 2.4 times in sporozoites, 1.6 times in merozoites, and 1.5 times in microgametes. The ultrastructural features of the attachment site of C. muris to the mucus cells were remarkably different from those of C. parvum and its closely related species. The anterior projection of the protozoa (C. muris), the outer aspect of which was surrounded by a thick filamentous process of the host cell, has not been reported at any developmental stages of C. parvum or its closely related species. The size of the oocysts of strain RN 66 was larger than that of Korean mice origin. The above results reveal that the large type Cryptosporidium of Korean mice origin is identified as Cryptosporidium muris and this type was named as C. muris (strain MCR).     

Experimental infection in mice of Cryptosporidium muris isolated from a camel.

Cryptosporidium muris-like oocysts in 5 g of camel feces were concentrated by the Sheather sugar flotation method, saline-washed, pelleted and reconstituted to an approximate concentration of 2.5 million per ml. One- to 2-microliters inocula were given per os to 25 2- to 20-day-old mice, with 15 contact control litter-mates. The 15- and 20- day inoculates had acid-fast-positive fecal smears by day-12 after inoculation. Necropsies of mice inoculated at 2 and 5 days of age showed colonization only of gastric glands by day-12, and day-27, respectively. Control mice were negative. Identical methods using similar oocysts from cattle produced no evidence of transmission.     

Freeze-fracture studies on tardigrade cuticle

The cuticles of the heterotardigrade Echiniscus testudo and the eutardigrades Macrobiotus hufelandi and Milnesium tardigradum have been studied using freeze-fracture technique. Most of the layers seen in conventional TEM micrographs can be visualized. There is no clear evidence that the trilaminar components of the cuticle such as the outer epicuticle and the tripartite layer separating epi- and intracuticle or procuticle (whose membranous origin has been suggested by previous authors) fracture like a lipid bilayer. Microfibres not resolved or only poorly resolved by TEM can be recognized in the procuticle of all three species. Obviously their visualization depends upon the fracture angle. In Echiniscus testudo and Milnesium tardigradum the intracuticle or at least parts of it show a wavy arrangement of microfibres. Parts of the ventral intracuticle of E. testudo fracture in an obviously non-random pattern revealing distinct sublayers.     

Recombinant thioredoxin peroxidase from Cryptosporidium parvum has more powerful antioxidant activity than that from Cryptosporidium muris

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-irradiation. In an animal study, more than 25kGy of γ-irradiation was necessary to eliminate C. parvum infectivity from mice. In contrast, Cryptosporidium muris (murine Cryptosporidium), which lives in stomach epithelium, lost its infectivity in mice with 1kGy of γ-irradiation. Recently, it was found that thioredoxin peroxidase was highly expressed in C. parvum oocysts irradiated with high doses of γ-irradiation. Therefore we hypothesize that antioxidant activity of the thioredoxin peroxidase is involved in the radioresistance of C. parvum. To verify this, thioredoxin peroxidases of C. parvum (CpTPx) and C. muris (CmTPx) were expressed in Escherichia coli cells, and their antioxidant activities were compared. Both CpTPx and CmTPx belong to the 2-Cys family of peroxiredoxins. Hydrogen peroxide consumption was approximately 2- to 12-fold greater in recombinant CpTPx (rCpTPx) than in recombinant CmTPx (rCmTPx) in the presence of 0.2mM dithioerythritol or glutathione (GSH), respectively. The peroxidase activity of rCpTPx was highly enhanced by GSH, but that of rCmTPx was not. The minimum dose of rCpTPx required to protect supercoiled plasmid DNA from damage by metal-catalyzed oxidation was only 12% of that required with rCmTPx. The results showed that rCpTPx has more powerful antioxidant activity than rCmTPx. Further investigations on the role of CpTPx in the radioresistance of C. parvum are warranted.     

Freeze-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

Freeze-Fracture Study of the Site of Attachment of Cryptosporidium muris in Gastric Glands

ABSTRACT The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Freeze-fracture studies on mitochondrial membranes of spermatocytes

Summary Separation of the two-folded lamina of the mitochondrial cristae occurs in mitochondria of spermatocytes and spermatids. Freeze-fracture exposes large areas of the inner and outer halves of the inner membrane. The surface of the outer half of the inner membrane is concave, with small numbers of intramembranous particles (IMPs). Its distinctive feature is the presence of protruding particles surrounding a pit. On the inner half of the inner membrane, there are large numbers of densely-packed, irregularly-distributed IMPs, among which regular pits are seen. Morphometric analysis and reconstructions suggest that these structures are channels in the mitochondrial membrane with an internal diameter of approximately 18 nm. It is uncertain whether such mitochondrial structures are confined to the spermatocyte or whether they may also occur in other cells.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Natural infection of Cryptosporidium muris (Apicomplexa: Cryptosporiidae) in Siberian chipmunks

Coprologic examination of nine Siberian chipmunks (Eutamias sibiricus) imported from Southeast Asia revealed infection with Cryptosporidium sp. Experimental inoculation of BALB/c mice proved their susceptibility to the infection. Infected mice shed oocysts 14-35 days postinfection. Oocyst morphology was similar to that reported for C. muris in previous studies, oocysts were 8.1 (7.0-9.0) x 5.9 (5.0-6.5) microns. Clinical signs were absent in naturally infected chipmunks and experimental mice. Histologic examinations of mice revealed numerous developmental stages of C. muris in the glandular stomach. Analysis of partial small subunit rRNA gene sequences confirmed identity of these isolates as C. muris. Our results represent the first report of C. muris in members of the family Sciuridae.     

In vitro culture of Cryptosporidium muris in a human stomach adenocarcinoma cell line

We investigated the optimal culture conditions for Cryptosporidium muris in a human stomach adenocarcinoma (AGS) cell line by determining the effects of medium pH and of selected supplements on the development of C. muris. The optimum pH of the culture medium required for the development of C. muris was determined to be 6.6. The number of parasites significantly increased during cultivation for 72 hr (p < 0.05) at this level. On the other hand, numbers decreased linearly after 24 hr of incubation at pH 7.5. When cultured in different concentrations of serum, C. muris in media containing 5% FBS induced 4-7 times more parasites than in 1% or 10% serum. Of the six medium supplements examined, only 1 mM pyruvate enhanced the number of C. muris in vitro. Transmission electron microscopic observation showed the developmental stages of C. muris in the cytoplasm of the cells, not in an extracytoplasmic location. The growth of C. muris in AGS cells provides a means of investigating its biological characteristics and of testing its response to therapeutic agents. However, a more optimized culture system is needed for the recovery of oocysts on a large scale in vitro.     

Freeze-fracture studies in the brown algaAsteronema rhodochortonoides

Summary The gross structure of the cell wall and the organization of the plasmalemma of the filamentous brown algaAsteronema rhodochortonoides were examined in replicas of freeze-fractured cells. The protoplasmic fracture face (PF) of the plasmalemma, apart from the single particles, exhibits two particular particle complexes, i.e., single linear arrays of closely packed particles, and well defined particle pentads. The former display a consistent relationship with the ends of microfibril imprints and therefore are considered as terminal complexes (TCs). They seem to be composed of subunits, each one consisting of two particles. The average diameter of the particles is 7 nm. The number of the subunits forming the TCs varies between 2 and 40. Short TCs, consisting of 3–5 subunits were also found on the PF of dictyosome vesicles, a fact suggesting the involvement of the Golgi apparatus in exocytosis of preformed TC portions. The occurrence, distribution and size of the TCs appear to be related to the developmental stage of the cell. A large number of TCs occur in actively growing cells, while a few or no TCs are found in differentiated cells. The pentads are rectangular structures consisting of five particles, four in the corners and one in the centre. Their dimensions are very constant, but their occurrence and distribution varies. They occur in young developing cells where TCs are few or absent, but were also observed in areas showing many TCs. In differentiated cells no pentads were found. Pentad-like structures were rarely observed on the PF of dictyosome vesicles or cisternae. The observations support the hypothesis that pentads are involved in the synthesis of matrix polysaccharides, which are the major components of brown algal cell wall and their synthesis begins before that of cellulose.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Freeze-fracture studies of annulate lamellae in zebrafish oocytes

Summary The zebrafish oocyte contains prominent stacks of annulate lamellae (AL) located primarily in a subcortical position of the ooplasm. Many lamellae comprising a stack eventually exhibit continuity with the rough-surfaced endoplasmic reticulum which is present in abundance in larger oocytes. Pore structure of both AL and nuclear envelope (NE) was studied and compared by use of freeze-fracture electron microscopy. In freeze-fracture replicas, the NE and AL pores were easily distinguished, and a variety of fracture planes with respect to the stacked AL were generated. The pore diameter of NE and AL is similar (100nm). The number of nuclear pores varied from an average of 40 pores/m² in early stage oocytes to nearly double this number in later stage oocytes. For AL, the center-to-center spacing (120–130 nm) and the number of pores per square micrometer (56–67) did not change markedly regardless of oocyte developmental stage examined. Hexagonal packing of AL pores is a common feature. The AL pores have an angular margin with octagonal symmetry suggested in some cases. The AL pore interior contains fibrillar and particulate components and, depending upon the fracture plane, may appear to be filled with a plug of material. Both P- and E-membrane fracture faces of AL have a relative scarcity of intramembranous particles. The non-porous membranes that extend from the AL, however, have a higher concentration of intramembranous particles.     

Freeze-fracture studies of the plasmalemma of Candida albicans after treatment with econazole-nitrate.

In electron microscopic studies the interior of the plasmalemma of Candida albicans was revealed by means of the freeze-fracture technique. The superficial structures of the extracellular (E) and protoplasmic (P) fracture faces differed negligibly from structures on the corresponding fracture faces of Saccharomyces cerevisiae. Following treatment with 2.2 x 10(-5) M econazole nitrate a layer, present on the P face in the form of a tight matrix of globular proteins, dissolved into isolated groups of particles whose globular elements sometimes formed hexagonal patterns. As the damage progressed, fissure-shaped membrane invaginations on the P face disappeared. Parts of the outer lipid layer of the plasmalemma were torn off the cell wall and adhered in fragments to the P face. The ultrastructural changes in the plasmalemma induced by econazole nitrate temporally correlate with an increase in the permeability of the cell envelope found in physiological studies performed by other authors.