Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy

Immune sera from mice immunized with the 143/140 kDa protein have been shown to partially block erythrocyte invasion by P. knowlesi merozoites. Therefore, immunoelectron microscopy utilizing ultracryomicrotomy, antibody to 143/140 kDa protein, and protein A gold particles were used to determine the precise localization of this protein in malarial parasites. Gold particles were not seen associated with young trophozoites but appeared in the parasite cytoplasm as the parasites grew to multi-nucleate schizonts. In presegmenter-schizonts, gold particles were associated with the well-developed endoplasmic reticulum, the parasite plasma membrane, and the parasitophorous vacuole membrane. The surface of merozoites was covered with gold particles. Maurer's clefts, which appeared in Plasmodium infected erythrocytes, were also associated with gold particles. These observations suggest that 143/140 kDa protective malarial proteins may be synthesized in the endoplasmic reticulum of P. knowlesi schizonts before being transported to the surface of the schizonts and merozoites. Shedding of the merozoite surface coat may be responsible for the presence of the 143/140 kDa proteins in the parasitophorous vacuole and Maurer's clefts.     

Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30-45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Invasion and Early Development of Sarcocystis muris (Apicomplexa,Sarcocystidae) in Tissue Cultures1

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30–45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Fine Structural Observations of the Erythrocytic Stages of Plasmodium chabaudi Landau, 1965*

SYNOPSIS. Electron microscopic examination of Plasmodium chabaudi in mouse erythrocytes revealed many characteristics resembling those observed in other mammalian malarial parasites. A double unit membrane surrounds the trophozoite cytoplasm which contains many ribonucleoprotein particles, a limited amount of endoplasmic reticulum and membraned organelles including sausage-shaped vacuoles and multilaminated membraned bodies. More or less circular double membraned vacuoles, possibly cross sections of the sausage-shaped vacuoles, are common. Typical protozoan mitochondria are lacking. The limiting membrane of the merozoites is triple-layered. Paired organelles and small dense bodies are found in the merozoites along with dense granular masses in the nuclei. Trophozoites have cytostomal structures as well as invaginations of the plasma membrane at sites where no cytostomes are evident. Digestion appears to occur in single membrane-bound vesicles which contain one to several pigment grains. P. chabaudi frequently contains multiple food vacuoles and has polymorphism manifested in part by the presence of cytoplasmic extensions and of nuclei with a variety of shapes. Several apparently free forms are noted, often accompanied by a thin rim of host cytoplasm. “Appliqué” forms are common among the trophozoites as are forms in which 2 or more trophozoites are joined together. Finally, alterations in the host cytoplasm resembling the socalled Maurer's clefts are frequent. Ferritin-containing vacuoles also appear in the host cell.     

Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.     

Ultrastructure of Babesia WA1 (Apicomplexa: Piroplasma) during infection of erythrocytes in a hamster model

Babesia Washington-1 (WA1) is a newly identified intraerythrocyte infectious agent of human babesiosis in the western United States. The purpose of the present study is to describe the ultrastructural changes in affected erythrocytes during the infectious process in a susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female hamsters were inoculated intraperitoneally (i.p.) with 1.8 x 10(9) Babesia WA1-infected erythrocytes originally isolated from a human case and serially passaged in hamsters. Saphenous vein blood samples (20 microl) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postinoculation (PI). Parasitemia was determined at each time interval by quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70, and almost 100% parasitemic erythrocytes at the corresponding PI time interval, respectively. Animals showed weakness and dehydration 72 hr PI inoculation, and were killed by 96 hr PI. Selected blood samples from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated in ethanol gradients, resin embedded, and then thin sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy or gold-coated for scanning electron microscopy (SEM). Shape and surface membrane changes in erythrocytes were demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a "swiss cheese" appearance 96 hr PI. Several erythrocytes had irregular surface membranes and Babesia WA1 organisms were seen at different stages of development within erythrocytes, from single trophozoites to several merozoites (young trophozoites), some of them dividing to form typical tetrads. In general, Babesia WAI induced severe morphological changes in the erythrocytes, and these changes were more evident in almost all infected cells 96 hr PI.     

Characterization of a membrane-associated rhoptry protein of Plasmodium falciparum

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.     

Etude Ultrastructurale des Mérozoites et de la Schizogonie des Coccidies (Eimeriina): Eimeria magna (Perard 1925) de I'Intestin des Lapins et E. tenella (Railliet et Lucet, 1891) des Coecums des Poulets

SYNOPSIS. An electron microscopic study is made of merozoites and schizogony of Eimeria magna and Eimeria tenella from rabbits and chickens infected 5 days before fixation.
The merozoite outer layer is formed by a unit membrane lined by a dense osmiophilic layer. A micropyle is present. The apical complex of the cell is constituted by a conoid surmounted by 2 rings and surrounded by another from which about 26 subpellicular, tubular fibrils start. Two "rhoptries" (= toxonemes) go thru the conoid to the apex of cell. Rare sarconemes (= convoluted tubes) are disseminated in the anterior part of merozoites. A nucleus with nucleolus, Golgi apparatus, mitochondria, endoplasmic reticulum, lipid globules and glucidic grains were observed.
Schizogony starts by the formation of a multinucleated schizont which has a centriolar structure. The new merozoites appear as evaginations of the schizont's membrane. Cellular organelles (conoid, rhoptries, micropyle, sarconemes) differentiate and the nuclei enter the diverticula of the schizont. Then the development of merozoites proceeds by "external budding".
The ultrastructural similarities between the merozoites of Eimeria and the endodyocytes of Toxoplasmea, appear to us to be extremely interesting and indicate a close relationship between the Toxoplasmea and the Coccidia.     

Vanadyl and vanadate inhibit Ca2+ transport systems of the adipocyte plasma membrane and endoplasmic reticulum

Vanadate and vanadyl have many insulin-mimetic effects on cellular metabolism and also have been shown to alter cellular Ca2+ fluxes. In this report, vanadate and vanadyl, like insulin, are shown to inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase/Ca2+ transport system as well as Ca2+ transport by endoplasmic reticulum from rat adipocytes. Ca2+ transport by the endoplasmic reticulum was inhibited half-maximally (I50) by vanadate and vanadyl at concentrations of 30 and 33 microM, respectively. Inhibition of the plasma membrane Ca2+ transport by vanadate and vanadyl was less sensitive, with I50 values of 144 and 92 microM, respectively. These I50 values for plasma membrane Ca2+ transport were similar when measured under conditions of calmodulin-stimulated and non-calmodulin-stimulated Ca2+ transport. The predominant effect of both ions on the kinetic parameters of Ca2+ transport was a substantial decrease in the Vmax by 43-46% for both transport systems. An increase in intracellular Ca2+ following the inhibition of the (Ca2+ + Mg2+)-ATPase/Ca2+ pump in the plasma membrane and endoplasmic reticulum by these vanadium ions may result, at least in part, in the observed insulin-mimetic alterations in cellular metabolism.     

Hexadecylphosphocholine interferes with the intracellular transport of cholesterol in HepG2 cells

We have shown, in a previous publication, that nontoxic concentrations of hexadecylphosphocholine exert an antiproliferative effect on HepG2 cells. Hexadecylphosphocholine also interferes with the biosynthesis of cholesterol and phosphatidylcholine. We have now extended our studies to try to establish the molecular mechanism by which hexadecylphosphocholine disrupts cholesterol homeostasis. Using radiolabelled substrates we determined the effect of hexadecylphosphocholine on cholesterol synthesis, the destiny of cholesterol from low-density lipoprotein and the transport of cholesterol between the plasma membrane and the endoplasmic reticulum. Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by western blotting and RT-PCR, respectively. HepG2 cells exposed to hexadecylphosphocholine showed an increase in cholesterol biosynthesis when acetate, but not mevalonate, was used as a substrate. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) and low-density lipoprotein receptor, as well as the corresponding mRNA expression, increased after 24 h of treatment with hexadecylphosphocholine. Cholesteryl linoleate in low-density lipoprotein uptake and further hydrolysis of these esters increased but the cholesterol esterification was reduced after 6 h of treatment with alkylphosphocholine. Cholesterol transport from the plasma membrane to the endoplasmic reticulum was impaired by hexadecylphosphocholine. In conclusion, hexadecylphosphocholine interfered with the transport of cholesterol from the cell surface to the endoplasmic reticulum, leading to a depletion of cholesterol in the endoplasmic reticulum and a deregulation of cholesterol biosynthesis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine synthesis produces an alteration in the phosphatidylcholine/cholesterol ratio that may well be responsible for the antiproliferative activity exhibited by hexadecylphosphocholine in HepG2 cells.     

Bovine babesiosis and anaplasmosis: Control by premunition and chemoprophylaxis

Experiments were carried out to evaluate two systems: (1) premunition and (2) chemoprophylaxis for the control of bovine babesiosis and anaplasmosis in the Cauca River Valley, Colombia. Control of these diseases was achieved by inoculating cattle with virulent Babesia bigemina, Babesia argentina, and Anaplasma marginale and subsequent treatment with Imidocarb and Gloxazone to moderate the postpremunition reactions. Chemoprophylactic treatment with Imidocarb and Gloxazone was administered to cattle before and during field exposure. Premunized cattle were highly resistant to tick-borne (Boophilus microplus) challenge. Imidocarb had therapeutic and chemoprophylactic properties against babesiosis, but appeared toxic. Gloxazone moderated the A. marginale postpremunition reaction, but failed to prevent clinical anaplasmosis under the conditions of this investigation.     

Differential time-course of induction of rat liver gamma-glutamyltransferase and drug-metabolizing enzymes in the endoplasmic reticulum, Golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two-dimensional electrophoresis

This study was conducted to follow as a function of time the activity of gamma-glutamyltransferase in the various membranes of rat liver cells after a single dose of phenobarbital (PB) (75 mg kg-1 body weight). Gamma-glutamyltransferase induction was maximal 24 h after PB treatment in both the rough endoplasmic reticulum and the plasma membranes. This pattern of induction differed from that of some drug metabolizing enzymes. While total cytochrome P-450 content was enhanced mainly in endoplasmic reticulum until 48 h after PB treatment, UDP-glucuronosyltransferase activity was not greatly altered by PB under the same conditions. The comparison of two-dimensional electrophoretic polypeptide profiles of each subcellular membrane isolated from control and phenobarbital-treated rats revealed important variations induced by PB. In plasma membranes, the heaviest subunit (apparent Mr = 60 x 10(3)) of hepatic gamma-glutamyltransferase was provisionally identified as a collection of polypeptide which differ only by their pI. The concentration of these polypeptides was smaller in the endoplasmic reticulum where they were of lower apparent molecular mass. This suggests that the gamma-glutamyltransferase precursor is already processed at the level of the endoplasmic reticulum but it is still not completely mature or glycosylated. Five days of continuous PB treatment induced by appearance of new gamma-glutamyltransferase isoforms in plasma membranes. We demonstrate that after a single injection of PB, gamma-glutamyltransferase activity increases simultaneously with some drug-metabolizing enzymes, such as total cytochrome P-450 but not with others, such as UDP-glucuronosyltransferases.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.     

A MORPHOMETRIC STUDY OF THE REMOVAL OF PHENOBARBITAL-INDUCED MEMBRANES FROM HEPATOCYTES AFTER CESSATION OF TREATMENT

It is well known that phenobarbital (PB) treatment produces an increase in the amount of cytoplasmic membranes of hepatocytes, with a parallel enhancement in the activity of drug-metabolizing enzymes. However, little is known about how the induced membranes are removed after the drug treatment is stopped. To consider this problem, the recovery of rat hepatocytes from PB induction (five daily injections, 100 mg/kg) was followed morphometrically. Treatment with PB produced a cellular enlargement (26%) due to increases in the volume of the cytoplasmic matrix (20%) and the volume (100%) and surface area (90%) of the smooth-surfaced endoplasmic reticulum (SER). The volume of the nuclei and the surface area of the Golgi apparatus were also increased, but no changes were detected in the volumes of the mitochondria or peroxisomes. The SER membranes induced by the PB were removed within 5 days after the end of the treatment period. During this period of membrane removal, we observed an increase in the volume (800%) and number (96%) of autophagic vacuoles without a change in dense bodies. A morphometric analysis of the content of the autophagic vacuoles showed that the endoplasmic reticulum membranes were preferentially removed, and from this we conclude that the formation of autophagic vacuoles was not a random process. Our findings show that the removal of excess cytoplasmic membranes is associated with an increase in autophagic activity and thus demonstrates the presence of a specific cellular mechanism which may be responsible for the bulk removal of PB-induced membranes.     

Giardia muris: ultrastructural analysis of in vitro excystation

Giardia muris cysts were examined by transmission electron microscopy before treatment, after induction, and at timed intervals during the incubation phase of in vitro excystation. Untreated G. muris cysts had a thick cyst wall composed of a fibrous outer wall and a thin, electron-dense inner membrane which extended from the trophozoite plasma membrane. The cytoplasm was devoid of endoplasmic reticulum, Golgi bodies,and mitochondria. Numerous large vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cysts. Following induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane bound vesicles were seen in the peritrophic space closely adhering to the surface of the trophozoite. These vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplasmic vacuoles but displayed well-developed ribbons of microtubular bodies, probably precursors of ventral disk, lateral flange, and median bodies and also contained extensive granular endoplasmic reticulum. No more than two nuclei were observed within each organism. The earliest excysted organisms were observed 0-5 min after incubation had begun and most organisms had excysted within 10 min. Cytokinesis occurred only after excystation was complete.     

Calreticulin, Ca2+, and calcineurin - signaling from the endoplasmic reticulum

Calcium (Ca2+) is a universal signalling molecule involved in many aspects of cellular function. The majority of intracellular Ca2+ is stored in the endoplasmic reticulum and once Ca2+ is released from the endoplasmic reticulum, specific plasma membrane Ca2+ channels are activated, resulting in increased intracellular Ca2+. In the lumen of the endoplasmic reticulum, Ca2+ is buffered by Ca2+ binding chaperones such as calreticulin. Calreticulin-deficiency is lethal in utero due to impaired cardiac development and in the absence of calreticulin, Ca2+ storage capacity within the endoplasmic reticulum and inositol 1,4,5-trisphosphate (InsP3) receptor mediated Ca2+ release from the endoplasmic reticulum are compromised. Over-expression of constitutively active calcineurin in the heart rescues calreticulin-deficient mice from embryonic lethality. This observation indicates that calreticulin is a key upstream regulator of calcineurin in Ca2+-signalling pathways and highlights the importance of the endoplasmic reticulum and endoplasmic reticulum-dependent Ca2+ homeostasis for cellular commitment and tissue development during organogenesis. Furthermore, Ca2+ handling by the endoplasmic reticulum has profound effects on cell sensitivity to apoptosis. Signalling between calreticulin in the lumen of the endoplasmic reticulum and calcineurin in the cytoplasm may play a role in the modulation of cell sensitivity to apoptosis and the regulation of Ca2+-dependent apoptotic pathways.     

Fine Structure of the Schizont and Merozoite of Isospora sp. (Sporozoa: Eimeriidae) Parasitic in Gehyra variegata (Dumeril and Bibron, 1836) (Reptilia: Gekkonidae)

SYNOPSIS. A study was made of the fine structure of some stages in the life cycle of an undesignated species of Isospora parasitic in a gecko. The merozoites which lay within a membrane-bound periparasitic vacuole in the host epithelial cell, had a striking similarity to Plasmodium, Lankesterella, Toxoplasma, Besnoitia, Sarcocystis, Eimeria and the M-organism. Each merozoite was invested with a triple-layered pellicle, the outer membrane of which was loosely applied. At the anterior end of the merozoite were conoid and apical rings; microtubules terminated in the posterior apical ring. Other organelles included nucleus, endoplasmic reticulum, mitochondria, micropyle, paired organelle, toxonemes and a variety of vacuoles. Although the sequence of development of the merozoite was not completely followed, some events in this process were recorded. The evidence suggests that anterior ends are formed early and that merozoites develop subsequently by a process of budding. The merozoite pellicle appears to be continuous with, altho structurally different from, the investing membrane of the parent cell.     

Vahlkampfia sp: structural observations of cultured trophozoites

Some structural observations on cultured Vahlkampfia sp. trophozoites are reported. Trophozoites are active and pleomorphic, producing large cell protrusions related to locomotion such as lamellipodia, filopodia and endocytic structures formed by hyaline cytoplasm, in which actin provides a framework that allows rapid changes in morphology. As observed by transmission electron microscopy, the cytoplasm is highly granular masking some cell organelles and the major cytoplasmic membrane systems. The structure of cell organelles such as the nucleus, endoplasmic reticulum, and digestive vacuoles is described. A common finding was the presence of 50 nm electron-dense round granules that are not limited by a membrane and that appear scattered in the cytoplasm, and whose function remains unknown. Apparently, the cell reserve material is glycogen, since complete trophozoites were positive to Schiff periodic-acid technique.