Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.     

Ultrastructural localization of prolactin-like antigenic determinants in neurosecretory cells in the brain of the honeybee (Apis mellifica).

With two different antisera to human prolactin (hPRL), the ultrastructural localization of PRL-like material in the bee brain is examined by means of the protein-A-gold method at the electron microscopical level. Labelling is found in electron-dense granules of medium size (150-200 nm in diameter) for the first time in insects. Such granules are distributed in the cytoplasm of the neurosecretory cells, their axons and their axon-terminals. The electron-dense granule is one criterion for identifying a neurosecretory cell. In the honeybee, hPRL-like material may serve as an old neurohormone with respect to its evolution.     

Effect of desferrioxamine and 2,2'-bipyridyl on the proliferation of Perkinsus atlanticus

Two types of iron chelators, desferrioxamine (DFO) and 2,2'-bipyridyl (BIP), selected for their differential binding properties, permeability and stoechiometry, were tested for their ability to inhibit the in vitro proliferation of the carpet shell clam parasite Perkinsus atlanticus. A tetrazolium-based assay was used to determine the effect of the drugs on cell proliferation. Both chelators were able to inhibit P. atlanticus proliferation in a dose-dependent manner, the 50% inhibitory concentration were 14 and 24 microM for DFO and BIP, respectively, in a 72 h test. This effect was reversed by co-addition of iron, confirming that this activity is due to the sequestration of iron. These results indicate a high degree of susceptibility of the protozoan parasite to chelator-induced iron deprivation. However, this effect was reversible upon removal of the drugs, indicating that the action of both chelators was cytostatic. For the range of concentrations tested the combined drug effects was not significantly higher than the additive effect of the individual drugs.     

Parasitism by the protozoan Perkinsus atlanticus favours the development of opportunistic infections

It has been suggested that opportunistic pathogens could contribute to the mortality of Perkinsus atlanticus-infected clams. Examination of Tapes semidecussatus clams from the northern Mediterranean coast of Spain revealed that while 86% of the clams heavily infected with P. atlanticus were co-infected by bacteria and/or viruses, neither non-infected nor lightly P. atlanticus-infected specimens had bacterial or viral infections. The bacteria, which had a Gram-negative cell wall, were always located in the apical pole of gill epithelial cells and enclosed within membranous compartments. Bacteria-containing cells were hypertrophied and showed dysplasia with loss of cilia and microvilli. The viruses shared ultrastructural, morphologic and cytopathic characteristics of a polyomavirus. Viral particles with icosahedral symmetry were found in both the cytoplasm and the nucleus of numerous cell types. Virus-infected cells showed severe alterations, including hypertrophy, reduction of the intracellular compartments and extrusion of the nuclear envelope. Moreover, gill epithelial cells showed disorganization and swelling of the apical region, which affected the ciliary structure. Our findings show that P. atlanticus parasitism favours the development of opportunistic infections which have detrimental effects in this clam population.     

Entamoeba invadens: Dynamics of DNA synthesis during differentiation from trophozoite to cyst

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0–8 h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8–24 h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24–72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.     

Phenoloxidase activity in three commercial bivalve species. Changes due to natural infestation with Perkinsus atlanticus

The phenoloxidase (PO) activity of the haemolymph and haemocytes from three clam species of commercial interest (Ruditapes philippinarum, Chamelea gallina and Tapes decussatus) has been compared. The activity was assayed spectrophotometrically by recording the formation of dopachrome from L-DOPA using sodium dodecyl sulphate, laminarin, trypsin or lipopolysaccharide as elicitors. Fewer PO units were observed in the haemolymph from T. decussatus than in the haemolymph from R. philippinarum, while the highest values were found in C. gallina. In all cases the activity was only significantly increased when sodium dodecyl sulphate was used as elicitor. PO activity in the haemocytes of all three clam species showed a very similar pattern to that found in the haemolymph from the same species. Furthermore, T. decussatus naturally parasitized by Perkinsus atlanticus (Protozoa, Apicomplexa) was used to study the influence of such infestation on PO activity, which was found to increase significantly in both haemolymph and haemocytes compared with non-infected (control) samples. PO activity in the haemocytes and in the haemolymph was higher when the level of parasitization was low or medium, respectively, and SDS was used as elicitor. No statistically significant differences were observed when the parasitization level was high. The present work constitutes the first report on the influence of this parasite on PO activity in haemolymph and haemocytes from T. decussatus.     

Molecular karyotype analysis of Perkinsus atlanticus (Phylum Perkinsozoa) by pulsed field gel electrophoresis

Perkinsus atlanticus is a pathogenic protist that infects the clam Ruditapes decussatus. Although it was recently proposed that the genus Perkinsus belongs to a new phylum, Perkinsozoa, in the infra-kingdom Alveolata, there remain different opinions about whether this genus should form a phylum on its own and consequently divergent views about its taxonomic characterization. In this work, we have identified nine chromosomes by pulsed field gel electrophoresis (PFGE) combined with densitometry analysis. The obtained karyotype of Perkinsus atlanticus, like that of other early branches of the dinoflagellate lineage, displays a more conventional chromosome organization, different from that of most dinoflagellates.     

Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.     

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (< 1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin.     

Ultrastructural differentiation during drosophila neurogenesis in vitro

Drosophila melanogaster neuroblasts differentiate in vitro, and each gives rise to a cluster of about 18 daughter neurons. Electron microscopic observations of single clusters show that axons from daughter neurons form a neuropile within the cluster of cell bodies. The neuropile increases in size and complexity for several hours, during which time chemical, and probably electrotonic, synapses form between neurites. Clear vesicles with diameters of about 35 nm and dense core vesicles with diameters of about 60 and 160 nm were detected. The development of the neuropile indicates that the prerequisite cell recognition phenomena were manifested during differentiation in vitro, and the complexity of the neuropile suggests it may have attained the capacity to process information.     

Ultrastructural localization of wheat germ agglutinin-binding sites on surfaces of chick embryo cells during early differentiation.

The objective of this work was to examine changes in a surface component of cells from the chick embryo during morphogenetic migrations of gastrulation. Two electron microscope techniques were used to localize cell-bound wheat germ agglutinin (WGA), a lectin which specifically binds N-acetyl glucosamine residues. One technique involved conjugation of peroxidase to WGA before reaction with the cells; the other technique used glucose oxidase to mark WGA which was already cell-bound. In both cases, binding was revealed using diaminobenzidine. Before formation of the primitive streak, all surfaces of the two-layered embryo bound WGA. After migration of cells through the streak, to form the three-layered embryo, not all cell surfaces bound WGA equally. Epiblast cells generally bound WGA lateral to the primitive streak but not during passage through the streak. Mesenchyme cells, after passage through the streak, bound WGA increasingly as they migrated away from the streak. A WGA-binding matrix was observed in the vicinity of the mesenchyme cells and on the dorsal surface of the endoblast. The ventral surface of the endoblast bound the lectin very poorly. In some instances, a peroxidase reaction product was consistently seen on certain surfaces which was not removable by addition of the simple hapten N-acetyl glucosamine. In these cases, the density of the deposit was lessened by use of diacetyl chitobiose as a hapten. This result, together with the reduction of reaction product following certain hyaluronidase treatments, suggests that WGA may be binding to hyaluronic acid as well as membrane glycoproteins.     

Immunocytochemical localization of mouse alpha 2-macroglobulinlike antigenic determinants on Schistosoma mansoni adults.

Antiserum prepared in rhesus monkeys against purified mouse alpha 2-macroglobulin (alpha2M) was labeled with peroxidase and incubated with both living and formalin-fixed S. mansoni adults (perfused from mice or rhesus monkeys) in order to test for the presence of mouse alpha2M antigenic determinants on their surfaces. Following standard cytochemical processing with the appropriate controls, adult worms of both murine and primate origin were found to have mouse alpha2M-like determinants on their surfaces. Earlier observations by other methods on the presence, approximate distribution, and quantitative difference of alpha2M antigenic determinants on adult worms of mouse or rhesus origin were confirmed.     

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro

Summary Cultures of embryonicDrosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. InDrosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in post-embryonic insect or vertebrate material. Mrs. Pilar Toribio-Fiorio provided excellent technical assistance, and Patricia Minter, the secretarial expertise. This investigation was supported, in part, by NIH Grant NS9330 and the James Douglas Research Fund to Robert L. Seecof and NIH Grant No. 1 RO1 CA17223-01 to Raymond L. Teplitz.     

Assignment of antigenic determinants to separated I-A kappa chains

The alpha- and beta-chains of the I-A kappa antigen from the AKTB-1b B cell lymphoma were separated by ion-exchange chromatography on CM-Sephadex in the presence of propionic acid and urea. Removal of the denaturants by dialysis produced isolated chains that regained a significant amount of their native configuration. These materials were used with a battery of monoclonal antibodies in a direct binding assay to localize specific alloantigenic determinants to the A alpha kappa or A beta kappa chains. This method allowed the assignment of the nominal specificity Ia. 17 and at least one epitope of the specificity Ia.2 to the A beta kappa chain. Finally, the I-A kappa antigen from the B cell lymphoma AKTB-1b was shown to be identical, by the criterion of tryptic peptide analysis, to that derived from normal B10.BR splenocytes. This constitutes the first demonstration that the polypeptide portion of a tumor-derived class II MHC antigen is identical to that derived from a normal tissue.     

Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay

The rRNA locus of Perkinsus atlanticus from the clam Ruditapes decussatus cultivated on the Atlantic coast of Spain was cloned and sequenced. Sequences of the internal transcribed spacer (ITS) from the rRNA locus were compared to sequences reported earlier for a P. atlanticus isolate from Portugal and to those from other Perkinsus species. The ITS I sequence of the Spanish P. atlanticus isolate was identical to the Portuguese P. atlanticus sequence and had 76.6% identity to the ITS1 of Perkinsus marinus. The ITS2 sequence had 99.7% identity to the Portuguese P. atlanticus ITS2, 92.5% identity to the P. marinus ITS2, and 99.5% identity to the Perkinsus olseni ITS2. We report for first the time the small subunit (SSU) and nontranscribed spacer (NTS) of P. atlanticus. The P. atlanticus SSU sequence was 99.6% identical to that of an unidentified Perkinsus species from the Australian clam Anadara trapezia and 98.0% identical to that of P. marinus. Further, our results support the proposal that P. atlanticus, P. olseni, and the Perkinsus sp. from A. trapezia constitute a subgroup of Perkinsus species distributed in the Pacific and eastern Atlantic, different from P. marinus that is distributed along the western edge of the Atlantic. Based on the NTS sequence of P. atlanticus from Spain and the differences with P. marinus NTS (62.2% identity), we developed a polymerase chain reaction (PCR)-based diagnostic assay with a lowest limit of detection of 0.01 amol of cloned NTS DNA as assessed on ethidium bromide-stained agarose gels. Specificity of the PCR-based assay was tested with samples from the clams R. decussatus, Ruditapes philippinarum, and Venerupis pullastra collected in P. atlanticus-enzootic areas of Spain. The specificity and sensitivity demonstrated for this NTS-based PCR assay validate its use as a tool for assessment of P. atlanticus in molluscs.     

Cognitive features of continuous antigenic determinants

We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes within 63 well-defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each epitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined form with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.