In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

ABSTRACT. Perkinsus marinus , a pathogen of eastern oysters ( Crassostrea virginica ), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus -1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue-black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus -1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus -1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell-free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.     

Use of a Tetrazolium-based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation

ABSTRACT. Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus . at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.     

Lipid Class and Fatty Acid Composition of the Protozoan Parasite of Oysters, Perkinsus marinus Cultivated in Two Different Media

The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n-9), 20: (n-9), 18:2(n-6) and 20:4(n-6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post-inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n-6), 20:5(n-3), 22:2delta7,15, and 22:6(n-3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

Effect of Fetal Bovine Serum Glycoproteins on the In Vitro Proliferation of the Oyster Parasite Perkinsus marinus: Development of a Fully Defined Medium

ABSTRACT. The oyster parasite Perkinsus marinus replicates in our medium consisting of Dulbecco modified Eagle's medium: Ham's F12 nutrient mixture (1:1) supplemented with 1–5% fetal bovine serum, with a doubling time of 24 hours during the exponential phase of the culture. Fetal bovine serum concentrations above 5% dramatically reduced parasite proliferation in a dose-dependent manner. We tested the individual effects of the three major protein components of fetal bovine serum (fetuin, transferrin and albumin) on the replication of the parasite in a serum-free medium. At the concentrations tested, fetuin enhanced parasite growth, whereas albumin had a modest positive effect and transferrin was inhibitory. Proteolytic digestion of fetuin, strongly diminished its growth-enhancing properties, indicating that the overall glycoprotein architecture may be required for activity. On the contrary, desialylation of fetuin slightly enhanced its growth-promoting activity. The addition of fetuin at 1.7 mg/ml to the serum-free DME: Ham's F12 medium yielded growth rates that are comparable to those obtained with our standard culture methodology. This has resulted in a fully defined culture medium that will allow for a rigorous characterization of excretory/secretory products involved in modulating or blocking the host's humoral and cellular defense mechanisms.     

Use of micromanipulation and "feeder layers" to clone the oyster pathogen Perkinsus marinus

Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating "feeder layer" of P. marinus on a 0.45-microm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.     

The Distribution of Perkinsus marinus in Gulf Coast Oysters: Its Relationship with Temperature,Reproduction, and Pollutant Body Burden

Oysters from 48 Gulf of Mexico sites were examined for presence and infection intensity of the endoparasite, Perkinsus (= Dermocystidium) marinus (Mackin, Owen and Collier, 1950) as part of NOAA's Status and Trends Mussel Watch Program. Prevalence exceeded 75% at 25 sites. Infection intensity did not vary with sex or reproductive stage. Latitude, total polynuclear aromatic hydrocarbon (PAH) content and industrial and agricultural land use significantly affected the parasite's distribution. PAH and pesticide concentrations were latitudinally dependent, suggesting an impact of spawning frequency on uptake and depuration. P. marinus analysis complements the use of pollutant body burden for determining change in environmental quality because it responds differently than pollutant body burden to the biology and ecology of the oyster.     

Ultrastructural traits of apoptosis

This review summarizes original and literature data on changes in the ultrastructure of major cell organelles during apoptosis obtained by transmission electron microscopy. Organelles that make the most crucial contribution to the initiation of apoptosis: plasma membrane, mitochondria, proteasomes, Golgi apparatus, and endoplasmic reticulum, were of our prime attention. The nucleus and cytoskeleton that undergo essential changes, were considered as well. Special attention was paid to the data on ultrastructural changes in the cell organelles observed recently by electron microscopic tomography and correlative microscopy, in particular, to remodeling of mitochondrial crista junctions and microtubules during the execution phase of apoptosis.     

人胎盘滋养层细胞原代的体外培养与改进

目的:建立与改进纯度较高的适于实验研究的人绒毛膜滋养层细胞。方法:采用胰蛋白酶消化法消化人正常妊娠6～8周胎盘组织,以35%、45%2个Percoll密度梯度进行分离纯化,并用免疫组化及透射电镜等技术对其生物学特性、细胞内部结构进行观察。结果:胎盘组织中滋养层细胞角蛋白染色阳性,血管内皮细胞及基质成分波形蛋白染色阳性,经该法分离纯化的细胞角蛋白染色阳性者(滋养层细胞)占90%以上,透射电镜观察示所获细胞有典型滋养层细胞结构。结论:该法简便易行,可获得合乎实验要求的人滋养层细胞,可供后续实验研究。     

Serological affinities of the oyster pathogen Perkinsus marinus (Apicomplexa) with some dinoflagellates (Dinophyceae)

The protozoan oyster pathogen Perkinsus marinus is classified in the phylum Apicomplexa, although molecular-genetic and ultrastructural evidence increasingly concur on its closer phylogenetic relationship with the dinoflagellates. To test for evidence of serological epitopes common to P. marinus and dinoflagellates, we probed 19 free-living and 8 parasitic dinoflagellate, or dinoflagellate-like, species for cross-reactivity with polyclonal antibodies to P. marinus. Three of 19 free-living dinoflagellates (16%), and 7 of 8 parasitic dinoflagellates (88%) were labeled by anti-P. marinus antibodies. In reciprocal immunoassays using polyclonal antibodies to the Hematodinium sp. dinoflagellate parasite of Norway lobsters, Nephrops norvegicus, P. marinus and the same 7 parasitic dinoflagellates labeled by anti-P. marinus antibodies, were again labeled. The dinoflagellate-like parasite of prawns Pandalus platyceros was not labeled by either antibody reagent. These reciprocal results confirm the presence of shared antibody-binding epitopes on cells of P. marinus and several dinoflagellates. The apparent widespread serological affinity between P. marinus and the parasitic dinoflagellates suggests a closer phylogenetic link to the syndinean dinoflagellate lineage. The consistent failure of the dinoflagellate-like prawn parasite to bind either antibody reagent shows that this parasite is serologically distinct from both P. marinus and Hematodinium-species parasitic dinoflagellates.     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Ultrastructural study of the epidermis in two free-living Platyhelminthes, Mesostoma viaregginum and M. productum (Rhabdocoela, Typhloplanida)

Abstract. The epidermis of the free-living typhloplanids Mesostoma viaregginum and M. productum (Mesostominae) is described. In both species, the epidermis has polarized cells with nuclei located at the basal part of the cell, whereas mitochondria are in the apical one. The epidermis is entirely covered by microvilli and locomotory cilia anchored in the cytoplasm by vertical and horizontal rootlets. Rootlets exhibit distinct length and periodic structure in the two species. Furthermore, in each species vertical and horizontal rootlets possess different periodic structure. The pattern of termination of microtubules in epidermal cilia is described for the first time in the Typhloplanida; central microtubules shift along one axonemal side, doublets 1 and 6–9 lose their microtubule B, and gradually peripheral doublets become singlets. Finally, an electron-dense material caps the tip of the cilia. This pattern of termination closely resembles that of Temnocephalida, Kalytorhynchia, and Dalyelliida examined so far, but differences exist.     

Investigation into the Physiological Significance of the Phytohormone Abscisic Acid in Perkinsus marinus,an Oyster Parasite Harboring a Nonphotosynthetic Plastid

Some organisms have retained plastids even after they have lost the ability to photosynthesize. Several studies of nonphotosynthetic plastids in apicomplexan parasites have shown that the isopentenyl pyrophosphate biosynthesis pathway in the organelle is essential for their survival. A phytohormone, abscisic acid, one of several compounds biosynthesized from isopentenyl pyrophosphate, regulates the parasite cell cycle. Thus, it is possible that the phytohormone is universally crucial, even in nonphotosynthetic plastids. Here, we examined this possibility using the oyster parasite Perkinsus marinus, which is a plastid‐harboring cousin of apicomplexan parasites and has independently lost photosynthetic ability. Fluridone, an inhibitor of abscisic acid biosynthesis, blocked parasite growth and induced cell clustering. Nevertheless, abscisic acid and its intermediate carotenoids did not affect parasite growth or rescue the parasite from inhibition. Moreover, abscisic acid was not detected from the parasite using liquid chromatography mass spectrometry. Our findings show that abscisic acid does not play any significant roles in P. marinus.     

Alpha-synuclein,especially the Parkinson's disease-associated mutants,forms pore-like annular and tubular protofibrils

Two mutations in the alpha-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinson's disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of alpha-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of alpha-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type alpha-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of alpha-synuclein protofibrils. These structures may contribute to the pathogenesis of PD.     

Ultrastructural studies on the epithelia of the olfactory organ of cyprinodonts (teleostei,cyprinodontoidea)

Summary The epithelia of the olfactory organ of two cyprinodontoid fish species were studied both by transmission and scanning electron microscopy. The relatively flat floor of the organ is covered by sensory and nonsensory epithelia. The latter is distributed in the form of bands or ridges separating distinct areas of sensory epithelium. Differences between the olfactory organs of the two species investigated related only to the topography and quantitative distribution of the epithelia. Their ultrastructural features are very similar. The nonsensory stratified squamous epithelium contains numerous goblet cells and surface cells provided with microridges. A hypothetical function of the microridges is discussed. The sensory epithelium consists mainly of basal, supporting, and two types of sensory cells, i.e., ciliated and microvillous receptor cells. The cilia exhibit a predominant 9+0 microtubule pattern. Both epithelia are covered by a mucus layer in which all surface structures seem to be embedded. The possible nature, origin, and movement mechanisms of the mucus are discussed.This work was supported by the Deutsche ForschungsgemeinschaftDedicated to Prof. Dr. med. W. Bargmann on the occasion of his 70th birthday     

In Situ Embedding of Cell Monolayers Cultured on Plastic Surfaces for Electron Microscopy

Various procedures suitable for routine in situ embedding of cell monolayers were tested including: (1) the use of different Epon substitutes, (2) the use of different types of plas-ticware obtained from different sources, and (3) different methods of preparing capsules for sectioning. Different resins reacted differently with different plastics and type of preparation. Merck Epon substitute bound to most of the plastics tested. Ladd Epon substitute released cleanly from all plastics tested when a suitable method of preparation was used. The results show that for routine embedding of cell monolayers it is necessary to select an appropriate Epon substitute and method of preparation of capsules for the type of plasticware used. A routine method is described, with various alternative steps which can be applied when particular difficulties are encountered.     

Giardia lamblia: Ultrastructural Study of the In Vitro Effect of Benzimidazoles

ABSTRACT Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

Development of a Rapid Detection Procedure for Ctyptosporidium, Using In Vitro Cell Culture Combined with PCR

A detection, viability, and infectivity assay was developed for Cryptosporidiurn parvum. Oocysts or excysted sporozoites were inoculated onto monolayers of CaCo-2 cells grown on chamber slides. C. parvum infection was monitored by three methods: a) application of a fluorescein-labeled anti-sporozoite antibody; b) PCR of a heat-shock protein gene fragment; and c) detection of mRNA from the heat-shock protein gene by RT-PCR.