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The first ultrastructural description of the genus Placus is presented. Each somatic kinetosome has a cone-shaped axosomal plate, nonoverlapping postciliary microtubules, an anteriorly directed kinetodesmal fiber, and a radial ribbon of transverse microtubules, which extend laterally under the ciliary furrow and insert in the cortical ridge. A closed ring of paired kinetosomes encircles the cytostome. A brosse begins adjacent to the oral pairs and extends posteriorly for one-fourth to one-half the cell's length. Autapomorphies for Placus include bowling pin-shaped toxicysts extruded onto a distinct area of the cell surface immediately posterior to the brosse, and a brosse kinety consisting of a single row of paired cilia. Placus and its sister taxon Spathidiopsis both have spiraling kineties composed of single cilia inserted into the side of the ciliary furrow. Spathidiopsis can be distinguished from Placus because it has a brosse consisting of two kineties (i.e. one composed of paired cilia, the other of single cilia), a row of rod-shaped toxicyst-bearing palps extending around one side of the oral area and along the length of the brosse, and a mid-cell cortical inpocketing containing toxicysts and a segment of the brosse. A revised listing of species assigned to the family Placidae is given., (© 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.)

Spathidiopsis and Placus are the only two genera within the family Placidae. The family has been placed in the class Prostomatea and order Prorodontida because its members have somatic monokinetids with a radial transverse ribbon, a straight non-overlapping postciliary ribbon, and anteriorly directed non-overlapping kinetodesmal fibril, an apical cytostome lacking specialized oral cilia, a brosse, and toxicysts. To confirm the stability of this placement, ultrastructural morphology and small subunit rRNA gene sequences of Spathidiopsis socialis, Spathidiopsis buddenbrocki, and Placus striatus were determined. These data were combined with information from other ciliates, and phylogenetic trees were generated using maximum-likelihood and maximum-parsimony methods. The analyses confirmed the family Placidae to be a monophyletic group in the Prostomatea with the Placidae a sister group to a Cryptocaryon + Coleps + Prorodon clade., (© 2011 The Author(s) Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.)

The Synhymeniida is characterized both by a band of somatic dikinetids, the synhymenium, extending across the surface of the cell and by a ventral cell mouth lacking specialized feeding cilia but subtended by a well-developed cyrtos. The synhymeniids have been hypothesized to be members of the class Nassophorea but our previous ultrastructural study of the synhymeniid genus Zosterodasys did not show any clear synapomorphies that would permit definitive placement in the Nassophorea or as a sister taxon to any of the other ciliate groups possessing a cyrtos. In the present study, simultaneous analysis of morphological and small subunit rDNA molecular data indicates that the Synhymeniida are sister to the class Phyllopharyngea and that this clade is, in turn, sister to the remaining Nassophorea, although this result is sensitive to dataset inclusion and alignment parameters. While this suggests that taxa with a ventral cyrtos might be united into a named taxon (e.g. resurrecting the Hypostomata), additional data are needed to reach a definitive conclusion.

With the amount of available sequence data rapidly increasing, supermatrices are at the forefront of systematic studies. As an alternative to supertrees, supermatrices utilize a total evidence approach where different genes and other lines of data are merged into a single data matrix, which is then analyzed in an attempt to obtain the phylogeny that best explains the data. However, questions may arise when combining data sets in which one or more taxa do not have sequences available for each individual gene. Two possible solutions to this situation are to either leave all taxa separate and code unavailable sequences as missing, or to combine taxa at a level for which monophyly is assumed a priori. By reanalyzing the previous work of, we show that combining taxa may yield misleading results, i.e., hypotheses of relationships that are not supported by the underlying data.

Many members of the cnidarian subclass Zoantharia (sea anemones, corals, and their allies) pass through a larval stage with eight complete mesenteries and without posterior musculature. This larva is usually transient, developing into an adult with 12 or more mesenteries. The adults of one family of sea anemones, the Edwardsiidae, bear the larval number and arrangement of mesenteries and lack the pedal disc seen in other sea anemones. The morphology of the Edwardsiidae has been interpreted in a number of ways: (1) the Edwardsiidae are the most basal extant zoantharian, having diverged before the evolution of additional mesenteries and basal musculature; (2) they are relatively advanced sea anemones that have secondarily simplified because they burrow in sand or mud rather than attaching to a hard substrate; or (3) edwardsiids are derived anemones that have retained a juvenile morphology through paedomorphosis. Phylogenetic analyses of small subunit ribosomal gene sequences reveal that the Edwardsiidae are derived zoantharians, nested within sea anemones. None of the proposed explanations fully explain the edwardsiid's body plan; edwardsiid anatomy is a mosaic of retained primitive and derived features. The results of the present study provide insight into zoantharian phylogeny and illustrate how phylogenetic tests can be used to study the evolution of cnidarian body plans.

Objective: To determine the nucleotide sequence of the alphaIIb gene from canine platelet-derived cDNA., Animals: 3 adult dogs., Procedure: First-strand cDNA was prepared from total RNA isolated from canine platelets. The cDNA was amplified, using specific primers in polymerase chain reaction (PCR), and the nucleotide sequence was obtained from purified PCR products., Results: Except for the nucleotide at position 694, results of all sequencing reactions of alphaIIb were identical for canine platelet-derived cDNA. Canine alphaIIb had 3 fewer codons than alphaIIb of humans. The nucleotide and deduced amino acid sequences of full-length canine alphaIIb shared > or = 83% similarity with the sequences established for humans. Segments of canine alphaIIb nucleotide and deduced amino acid sequences were > or = 78% similar to alphaIIb associated with 7 functional domains (extracellular, transmembrane, cytoplasmic, and 4 calcium-binding domains) in humans, with the highest degree of similarity correlating with the sequences of the 4 calcium-binding domains. Amino acid residues associated with development of alloantibodies in humans (Met837, Val837, Ile843, Ser843) are not encoded by canine alphaIIb., Conclusions and Clinical Relevance: The nucleotide variation at position 694 of canine alphaIIb may represent a polymorphism. The species differences in the alphaIIb sequence may contribute to variations in receptor-li gand interactions. The high degree of alphaIIb sequence conservation of the 4 calcium-binding domains implies functional importance. Some disorders associated with alphaIIbbeta3 in dogs are clinically analogous to diseases in humans, and results indicate that dogs are an appropriate model for the evaluation of gene therapy and other treatments of platelet-associated disorders.

Glanzmann's thrombasthenia (GT) is an inherited, intrinsic platelet function defect that involves the platelet glycoprotein complex IIb-IIIa, also known as the fibrinogen receptor and the integrin alphaIIbbeta3. The defect was originally described by Dr. Glanzmann in humans in 1918 as a bleeding disorder that differed clinically from other known coagulopathies. Over the decades that followed, researchers determined the biochemical and molecular basis for the disease in humans. Otterhounds with thrombasthenic thrombopathia, described in the 1960s, were the only animal model that closely resembled the disease described in humans until 1996. At that time, a Great Pyrenees dog was identified with unequivocal clinical and biochemical features of Type I GT The cDNA encoding for glycoproteins IIb and IIIa were sequenced in normal dogs in 1999, allowing for identification of specific mutations causing Type I GT in both Otterhounds and Great Pyrenees dogs. Knowing the molecular basis for Type I GT in dogs as well as the cDNA sequences in normal dogs should enhance the understanding of structure/function relationships of the alphaIIbbeta3 integrin and provide an excellent animal model for studies aimed at correction of GT in humans. The following review focuses on the structure and function of this platelet receptor and reviews the molecular, biochemical, and clinical aspects of Glanzmann's thrombasthenia in humans and dogs.

Glannzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by qualitative or quantitative deficiencies of the platelet membrane glycoprotein alphaIIbbeta3. This is the first report of a molecular genetic basis for type I GT in dogs. As previously reported, a thrombasthenic Great Pyrenees dog (dog No. 1) experienced uncontrolled epistaxis despite results of coagulation screening tests, platelet quantitation, and von Willebrand factor quantitation that were within reference ranges. Platelet aggregation was minimal in response to agonists. Flow cytometry, autoradiography, and immunoblot experiments demonstrated either marked reduction or absence of glycoproteins alphaIIb and beta3. In this study, we report the presence of a 14-base insertion in exon 13 and defective splicing of intron 13 in the alphaIIb gene of two thrombasthenic dogs (Nos. 1 and 8). The insertion disrupted the fourth alphaIIb calcium-binding domain, caused a shift in the reading frame and resulted in a premature termination codon. Possible consequences of this mutation include decreased alphaIIb mRNA stability and production of truncated alphaIIb protein that lacks the transmembrane and cytoplasmic domains and a large portion of the extracellular domain. We identified the dam, sire, and three littermates of dog No. 8 as carriers of the alphaIIb mutation. Canine alphaIIb and beta3 genes share significant homology with the genes in human beings, making canine GT an excellent translational model for human GT. A defined molecular basis for canine GT will enhance ongoing gene therapy research and increase the understanding of structure-function relationships of this integrin.

The platelet glycoprotein complex alphaIIb beta3 is required for platelet-fibrinogen binding and platelet aggregation. This study was designed to characterize the nucleotide sequence of the canine platelet beta3 gene from cDNA. The nucleotide and deduced amino acid sequences of the canine beta3 gene were 92% and 96% homologous, respectively, with the sequences previously established for the beta3 gene of human beings. Within the beta3 gene, the nucleotide sequence of cDNA prepared from canine platelets shared homology of 89% for the cytoplasmic domain, 93% for the transmembrane domain, 92% for the extracellular domain, 94% for the arginine-glycine-aspartic acid (RGD) binding domain, and 97% for the region associated with Ca2+-dependent stabilization of the alphaIIb beta3 fibrinogen-binding pocket. The deduced amino acid sequence of canine beta3 was 100%, 97%, 96%, and 95% homologous with the cytoplasmic, transmembrane, extracellular, and RGD-binding domains, respectively, and was 100% homologous with the region associated with Ca2+-dependent stabilization of the alphaIIb beta3 fibrinogen-binding pocket of beta3 in human beings. The canine platelet cDNA signal peptide segment of the beta3 gene encodes for 22 amino acids, as compared with 26 amino acids previously reported for human beings. The deduced amino acid sequence of canine beta3 corresponds to the high-frequency allelic form for five of the six alloantigenic sites reportedly associated with human platelets: Leu33Leu40Pro407Arg489Arg636. The apparent amino acid residue in position 143 (Pen alloantigen) of canine platelet beta3 is histidine compared with arginine in human beings. Knowledge of the beta3 gene nucleotide sequence of normal dogs will facilitate the understanding of platelet alphaIIb beta3 structure-function relationships.

Sequences of the small subunit (SSU) ribosomal RNA are considered useful for reconstructing the tree of life because this molecule is found in all organisms and is large enough not to have become saturated with multiple mutations. However, these data sets are large, difficult to align, and have extreme biases in base compositions which makes their phylogenetic signal ambiguous. Large ambiguous data sets may have many most-parsimonious trees, and finding them all may be impossible using convential phylogenetic methods. To examine the reliability of the number and relationships of eukaryotic kingdoms proposed by previous analyses of the SSU, we calculated trees from aligned sequences from eukaryotes in the Ribosomal Database Project using parsimony jackknifing which uses a resampling procedure to rapidly search large data sets for the branches that are strongly supported and eliminates poorly supported groups. Two separate analyses were carried out: an analysis in which all bases were equally weighted, and one in which transversions only were used. The parsimony jackknife procedure was able to efficiently find trees in which most major groups of eukaryotes were supported and in which some evolutionary hypotheses proposed by previous workers were tested. The relationships of these major groups to each other were largely unresolved, indicating that the SSU data, as represented in this database, is insufficient for answering questions about these deep branches. Interestingly, the analysis of transitions differs from the results of the entire data set, primarily being less resolved. This indicates that transversional mutations are important contributors to the resolved structure of the tree.

Objective: To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation., Animals: 5 mature healthy horses., Procedure: Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 microM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody., Results: Released platelet proteins in the presence of beta-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean +/- SEM) 194 +/- 2, 159 +/- 2, 151 +/- 2, 104 +/- 2, and 95 +/- 1 kd. Immunoblots of released platelet proteins had a prominent 180 +/- 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 +/- 1 and 155 +/- 1 kd) were observed., Conclusions: Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets., Clinical Relevance: An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses.

Abstract- The order of states in a transformation series describes an internested set of synapomorphies. States adjacent to each other in the transformation series thus share a degree of homology not found in the other states. Whether the level of homology is relatively apomorphic is determined by rooting the order with outgroup comparison. The analysis of state order is a homology problem and is solved with a two-step process using similarity and congruence with other characters as criteria. Other methods that have been proposed (e.g. transformation series analysis, non-additive analysis, morphocline analysis, ontogenetic analysis) fail to apply both similarity and congruence, and thus cannot be used independently for determining character state order.

The superiority of cladistic methods to both synthetic and phenetic methods is briefly advanced and reviewed. Cladistics creates testable hypotheses of phylogeny that also give a highly informative summary of available data. Thus it best fits the criteria for a method for determining the general reference classification in biology. For protistologists in particular, cladistics is especially useful. Inundated by an abundance of ultrastructural, biochemical, and cell biological information, protistologists could be greatly helped by the informative way in which cladistics orders and summarizes the data. In addition to classifying protist taxa, hypotheses about the evolution of cell organelles and cellular could be scientifically formulated and tested by cladistics . Because cladistic classifications best summarize the data, they would also be best for making predictions about taxa and characters. They would, for the same reason, be the most stable. Widespread adoption of cladistic methods would serve to stabilize the now fluid state of protist taxonomy. It is for all of these reasons that such methods best suit the needs of the evolutionary protistologist .

A benthic marine protist (Stephanopogon) with a homokaryotic nucleus has long been considered to be a gymnostome ciliate. It has been important in hypotheses concerning the origin of ciliates, the evolution and origin of the dual nuclear apparatus of contemporary species of the Ciliophora, and the origin of the multicellular Eumetazoa. Ultrastructural observations reveal that the organism should be reclassified as a flagellate, despite its superficial resemblance to ciliates.

Abstract- Cladistic analysis of data from ultrastruetural and other cell biological studies indicates that neither the classical two kingdom nor the commonly accepted five kingdom classifications accurately represent the cellular diversity of eukaryotes. The resulting cladogram indicates instead that there are nine major groups of eukaryotes., (© 1985 The Willi Hennig Society.)