The author describes the serendipitous discovery, conception, development, and history of Nissenbaum's Fixative while an undergraduate biology major in the early 1950s. The subsequent uses, applications, and modifications over the past forty-seven years are also described. Some of the modifications omitted from his short original paper are mentioned. Highlights of his subsequent career in the field of medicine are noted.

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites.

 Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC₄(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H₂O₂ and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgalactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.

Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-d-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by “cyst wall synthase” (β 1 ;ra 3 GalNAc transferase) into a highly insoluble β 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.

Trypanosoma cruzi trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that the release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.

We established monoclonal in vitro cultures of a Perkinsus sp. isolated from the baltic clam Macoma balthica and compared morphological features of various life stages by light and transmission electron microscopy to those of the currently accepted Perkinsus species: Perkinsus marinus, Perkinsus olseni, Perkinsus atlanticus, and Perkinsus qugwadi. Except that trophozoites were slightly larger than those of P. marinus, and that they underwent zoosporulation in culture, observation of our isolate under light microscopy did not reveal striking differences from any Perkinsus species. Perkinsus sp. from M. balthica shared fine structural characteristics with other Perkinsus species that clearly place it within this genus. Although zoospores of Perkinsus sp. from M. balthica were slightly smaller than those from other species, the ultrastructural arrangement and appearance of the apical complex and flagella seem to be identical to those of P. marinus and P. atlanticus. Our isolate also appeared, in some sections, to have cortical alveolar expansions of the plasmalemma at regions other than the anterior end and lobulated mitochondria that were reported as unique for P. qugwadi. Little consensus exists among authors in the assignment of taxonomic weight to any particular morphological feature to designate Perkinsus species. The present study of gross morphology and ultrastructure was complemented with molecular studies reported elsewhere, which propose that Perkinsus sp. from Macoma balthica is a distinct species.

A Perkinsus species was isolated from the baltic clam Macoma balthica and an in vitro culture established under conditions described for P. marinus. As reported previously, morphological features remarkable enough to clearly indicate that this isolate is a distinct Perkinsus species were lacking. In this study, regions of the rRNA locus (NTS, 18S, ITS1, 5.8S, and ITS2) of this isolate were cloned, sequenced, and compared by alignment with those available for other Perkinsus species and isolates. Sequence data from the rRNA locus and species-specific PCR assays indicated not only that Perkinsus sp. from M. balthica was not P. marinus, but it was different from P. atlanticus and P. olseni. The degree of difference was comparable to or greater than differences between accepted Perkinsus species. In particular, NTS sequence and length were dramatically different from that of P. marinus and P. atlanticus. Therefore, we formally propose to designate the Perkinsus sp. from M. balthica as a separate species, P. andrewsi n. sp. Primers based on P. andrewsi NTS sequence were used to develop a PCR-based diagnostic assay that was validated for species-specificity and sensitivity. PCR-based assays specific for either P. andrewsi or P. marinus were used to test for their presence in bivalve species sympatric to M. balthica. Although isolated from M. balthica, P. andrewsi was also detected in the oyster Crassostrea virginica and clams Macoma mitchelli and Mercenaria mercenaria, and could coexist with P. marinus in all four bivalve species tested.

We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas-specific molecular markers based on spliced-leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.

Morphogenetic processes during division in Uroleptus caudatus (Stokes, 1886) are described using protargol impregnation. As is typical for the family Urostylidae Bütschli, 1889, zigzag midventral cirri develop, but contrary to most species of this family the adoral membranelles of the proter are not renewed during division and two dorsomarginal kineties develop. These two atypical features occur in only two other species of the Urostylidae (Uroleptus musculus, Holosticha diademata), and in all species of the family Parakahliellidae Eigner, 1997, and in many of the Oxytrichidae Ehrenberg, 1838. Membranelles are also not renewed in the Orthoamphisiellidae Eigner, 1997. This shows that these three atypical members of the family Urostylidae are those which are most closely related to the other three hypotrichous families named above. The highly distinct zigzag midventral cirri clearly separate the family Urostylidae from the other three hypotrichous families. These cirri are used together with new morphogenetic features for a revised family diagnosis. The analysis of the Urostylidae revealed that during division all species of the family Urostylidae develop their cirral patterns, including the two rightmost ventral anlagen, in separate areas for each proter and opisthe. Thus, &ldquo≶ng primary primordia” are absent in the Urostylidae. This is similar to the morphogenetic pattern by which the family Parakahliellidae is defined (&ldquo≠okinetal 1”) indicating that the Urostylidae are more closely related to the Parakahliellidae than to either the Oxytrichidae or to the Orthoamphisiellidae. All 68 detailed descriptions of divisional morphogenesis in species of the Hypotrichida are analyzed now in this and two former papers. As a result all these species can be assigned or at least recognized to be closely related to one of the following four families: to the Orthoamphisiellidae (“within-row” anlagen), to the Oxytrichidae (&ldquo≠okinetal 3” anlagen), to the Parakahliellidae (&ldquo≠okinetal 1” anlagen), and to the distinctly different Urostylidae (&ldquo≳dventral” anlagen). Thus, all Hypotrichida can most probably be assigned after morphogenetic investigation to one of the four families.

We have isolated and characterized the micronuclear gene encoding the regulatory subunit of cAMP-dependent protein kinase of the ciliated protozoan Euplotes octocarinatus, as well as its macronuclear version and the corresponding cDNA. Analyses of the sequences revealed that the micronuclear gene contains one small 69-bp internal eliminated sequence (IES) that is removed during macronuclear development. The IES is located in the 5′-noncoding region of the micronuclear gene and is flanked by a pair of tetra-nucleotide 5′-TACA-3′ direct repeats. The macronuclear DNA molecule carrying this gene is approximately 1400 bp long and is amplified to about 2000 copies per macronucleus. Sequence analysis suggests that the expression of this gene requires a 1 ribosomal frameshift. The deduced protein shares 31% identity with the cAMP-dependent protein kinase type I regulatory subunit of Homo sapiens, and 53% identity with the regulatory subunit R₄₄ of one of the two cAMP-dependent protein kinases of Paramecium. In addition, it contains two highly conserved cAMP binding sites in the C-terminal domain. The putative autophosphorylation site ARTSV of the regulatory subunit of E. octocarinatus is similar to that of the regulatory subunit R₄₄ of Paramecium but distinct from the consensus motif RRXSZ of other eukaryotic regulatory subunits of cAMP-dependent protein kinases.

The flagellar apparatus of Ploeotia costata Farmer and Triemer was reconstructed using serial sectioning and TEM. The flagellar apparatus is similar to other euglenoids having two flagella arising from basal bodies connected by a striated fiber, and three asymmetrically arranged roots. The flagella emerge subapically from between the two ventral pellicle strips. The dorsal flagellum is 1/2 the body length and actively pulls the cell, while the ventral flagellum is twice the body length and drags along the substrate surface. The ventral and dorsal roots are on the opposite sides of their respective basal bodies, while the intermediate root is associated with the ventral flagellum on the side closest to the dorsal basal body. The dorsal root lines the dorsal side of the reservoir and after giving rise to the dorsal band lines the right side of the reservoir/canal. The ventral and intermediate roots join at the reservoir forming the intermediate-ventral root, which lines the left and ventral sides of the reservoir/canal. There was no evidence of a microtubule-reinforced pocket in P. costata. Comparisons with Ploeotia vitrea, Lentomonas applanatum, and related flagellar apparatuses led to the conclusion that the basic euglenoid flagellar structure is symplesiomorphic but with enough variation to be taxonomically diagnostic.

The trichites of Strombidium and related genera have been considered either as a cytoskeletal armature or as extrusomes. To demonstrate their true nature, a study was undertaken on two marine Strombidium species by ultrastructural and cytochemical analysis as well as in vivo experiments. Trichites, extending from the cortex into the cell, are rod-shaped, membrane-bounded, and have a complex structure. The following elements of the trichites, are distinguishable: an electron-transparent lumen, a laminated layer, and a compact layer. In trichites of one species, thin “rings” surround the lumen. Numerous short, curved tubules with a polysaccharide wall are present in the cytoplasm surrounding the trichites. At the cortical end, each trichite is enveloped by a &ldquo@p” of electron-dense proteinaceous material. In some cases, the cortical alveoli appear interrupted, forming a “hole” for trichite ejection. Ejection of rod-shaped structures, up to 5 times longer than resting trichites, was obtained by in vivo treatments with dextran and aminoethyldextran. Negative staining indicated that these structures were transformed trichites. As no other possible extrusive structures were observed in the cytoplasm of Strombidium, trichites were considered extrusomes.

We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activity is dependent on Ca² and Mg² ions, its basal activity is stimulated by phosphatidyl-inositol-4,5-bisphosphate (PIP₂), and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3-fold about 5 min after an abrupt decrease in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5-fold in response to an abrupt increase in osmolality to 617 mOsM. Cells grown for > 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions, indicating an adaptation to long-term exposure to hypo- or hyper-osmolarity. Immunologically, two isoforms, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti-PLD immunocomplexes revealed that either hypotonic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed increase when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isoform, suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.

Development of Leucocytozoon smithi during gametogenesis, fertilization, and ookinete differentiation was studied by light and electron microscopy. Gametogenesis occurred rapidly, within 1–2 min after gametocytes were ingested by black flies. Usually one axoneme, but not infrequently two, was observed in microgametes. The macrogamete nucleus was characteristically elongated and fragmented, with a convoluted nuclear envelope. Fertilization occurred within five min after ingestion of gametocytes by the vector. The entire axoneme and nucleus of the microgamete entered the cytoplasm of the macrogamete. Zygote differentiation resembled sporozoite formation in that a thickened inner membrane and subpellicular microtubules developed beneath the plasmalemma, followed by cytoplasmic protrusion or evagination to form the anterior end. Extension of the inner thickened membrane continued as the zygote elongated. Development of sausage-shaped ookinetes was completed within 6–8 h after ingestion of a blood meal by a black fly. Mature ookinetes possessed a single nucleus, double-layered pellicle, canopy, apical pore, polar ring complex, subpellicular microtubules, micronemes, crystalloids, abundant mitochondria, endoplasmic reticulum, and ribosomes. Comparison of development of L. smithi with species of Plasmodium and Haemoproteus revealed general similarities in both sexual and asexual development within the insect vector. A diagram summarizing life cycle events for L. smithi is included.