Ecology of testate amoebae (Protozoa: Rhizopoda) on peatlands in western Russia with special attention to niche separation in closely related taxa.

Testate amoebae (Protozoa: Rhizopoda) are frequently used as indicators of past environmental changes, and the interpretation of fossil assemblages depends upon our knowledge of ecological affinities of taxa in modern environments. A variety of taxonomic approaches have been used in fossil studies, mostly involving grouping of closely related taxa. This paper presents data from peatlands in western Russia relating surface wetness parameters to species occurrence. Relationships between species abundance, water table depth and soil moisture are modelled using weighted averaging, and species niches are calculated as optima and tolerance for these parameters. Niche separation of closely related taxa is examined in detail and it is shown that there is often a gradient of hydrological preference within each group of taxa. Wet to dry gradients include those found in the Trigonopyxis arcula group (T. arcula var. major > T. arcula > T. minuta), the Assulina-Valkanovia group (A. seminulum > A. muscorum > V. elegans), and the Trinema lineare group (T. lineare var. truncatum/ T. lineare > T. lineare var. terricola), all of which are associated with a large to small size gradient. In addition, spined forms within the Euglypha and Placocista genera are shown to consistently occur in wetter habitats than glabrous forms or those with shorter spines. It is concluded that palaeoecological studies should attempt the lowest taxonomic divisions possible within these groups, to maximise the ecological indicator value of the assemblages recorded.     

Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm

Long-chain fatty acids can transfer passively across mammalian cell membranes. However, under physiological conditions of low fatty acid to albumin ratios in the circulation, the major fraction of uptake appears to be mediated by a saturable, protein-facilitated component. A simple diffusion process becomes significant at high molar ratios of fatty acid to albumin as the concentration of free fatty acid in solution is increased. Identification of the mammalian membrane fatty acid transporter(s) has been the focus of active investigation by several research groups. In this review we discuss three candidate proteins: FABPm, FAT/CD36 and FATP which have been cloned and are currently being characterized. Recent evidence arguing for an important role of the fatty acid transport step in general metabolism and linking these proteins to physiologic or metabolic abnormalities is described.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Keystone Symposia on Molecular and Cellular Biology: ‘The molecular basis of cancer’: Organizers: Carol L. Prives and George F. Vande Woude,Taos, NM, 15–21 March 1999

The Keystone Symposium on the Molecular Basis of Cancer was an excellent meeting, which stimulated the exchange of a great deal of information. This report was prepared to organize some of the results that provided new insights into the regulation of cell proliferation and apoptosis. We were unable to report on all of the talks and posters due mostly to our limited capacity to absorb and digest the large amount of results presented at the meeting. We apologize to those whose results were not covered in this report.     

Phylogenetic affinities of Diplonema within the Euglenozoa as inferred from the SSU rRNA gene and partial COI protein sequences

In order to shed light on the phylogenetic position of diplonemids within the phylum Euglenozoa, we have sequenced small subunit rRNA (SSU rRNA) genes from Diplonema (syn. Isonema) papillatum and Diplonema sp. We have also analyzed a partial sequence of the mitochondrial gene for cytochrome c oxidase subunit I from D. papillatum. With both markers, the maximum likelihood method favored a closer grouping of diplonemids with kinetoplastids, while the parsimony and distance suggested a closer relationship of diplonemids with euglenoids. In each case, the differences between the best tree and the alternative trees were small. The frequency of codon usage in the partial D. papillatum COI was different from both related groups; however, as is the case in kinetoplastids but not in Euglena, both the non-canonical UGA codon and the canonical UGG codon were used to encode tryptophan in Diplonema.     

Characterization of Hemiselmis amylosa sp. nov. and phylogenetic placement of the blue-green cryptomonads H. amylosa and Falcomonas daucoides

The morphology and ultrastructure of a new freshwater blue-green cryptomonad, Hemiselmis amylosa sp. nov., is described. In addition, a marine blue-green cryptomonad isolate was confirmed as Falcomonas daucoides by electron microscopy and phycobilin analysis so that it could be included in molecular sequence studies, since the original isolate is no longer available. Complete ssu rRNA gene sequences for H. amylosa and F. daucoides were obtained. Our freshwater isolate of Hemiselmis possesses the same general features described for blue-green marine species, but it differs in having an eyespot, and multiple, single thylakoids penetrating the pyrenoid; therefore, a new blue-green, freshwater species is described. Phylogenetic analyses of H. amylosa and F. daucoides, as well as 24 other cryptophyte algae, indicate a monophyletic origin for all blue-green cryptomonads. Falcomonas forms a sister clade to blue-green cryptomonads, indicating that it is the most primitive extant blue-green cryptomonad and probably diverged early from other blue-green genera. Furthermore, we suggest that the eocyte blue-green cryptomonad may have originated from a Proteomonas-like progenitor that underwent a pigment change, resulting in a Falcomonas-like cell. Based on comparative morphology, the Proteomonas haplomorph may be a likely candidate in the evolutionary transformation from red to blue-green in cryptomonads; however, phylogenetic analyses neither support nor refute this hypothesis. Finally, the current status of cryptomonad classification is addressed.     

Systematics of the enigmatic kathablepharids, including EM characterization of the type species, Kathablepharis phoenikoston, and new observations on K. remigera comb.nov

The colorless flagellate Kathablepharis has consisted of five species based on light microscopic studies, and the ultrastructure of the type species, Kathablepharis phoenikoston, is described for the first time. The heterotrophic, marine flagellate Leucocryptos consisted of two species, but additional ultrastructural details for one of these, Kathablepharis remigera comb. nov. (= Leucocryptos remigera V?rs), indicates that it should be transferred to Kathablepharis. The cellular structure of these two species is similar to previously studied kathablepharids. However, there is variation in the feeding apparatus and cytoskeleton. The feeding apparatus of both species has a cytostome, a cytostomal ring, and cytopharyngeal rings. The cytoskeleton consists of inner microtubular arrays and outer or sub-pellicular microtubular arrays. In addition, several features of the flagellar apparatus are described for K. phoenikoston and K. remigera. The ultrastructure of these two species is compared with other kathablepharids to evaluate their taxonomy and phylogeny. We classify Kathablepharis and Leucocryptos in the family Kathablepharididae incertae sedis.     

Palmitate-induced apoptosis in cardiomyocytes is mediated through alterations in mitochondria: prevention by cyclosporin A

Palmitate, a C16 fatty acid found in high concentrations in the blood in acute myocardial infarction, induces apoptotic cell death. To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic chick heart and were treated with palmitate. Concentration-dependent loss of cell viability was established by loss of the ability of palmitate-treated cells to exclude propidium iodide (PI), metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and retain fluorescein diacetate (FDA). Dual staining with PI and FDA and subsequent analysis by FACS established that palmitate-induced cell death was predominantly necrosis whereas apoptosis occurred in 13% of all dead cells. The low proportion of palmitate-induced apoptosis was confirmed by evaluation of the DNA content or PI fluorescent staining of the DNA of permeabilized cardiomyocytes. A critical role for mitochondria in the pathogenesis of palmitate-induced cell death was demonstrated, for the first time, based on palmitate-induced reduction of mitochondrial activity as assessed by the mitochondrial-selective dye chloromethyl-X-Rosamine and the presence of a greater amount of the mitochondrial marker cytochrome C in the cytosol of palmitate-treated cardiomyocytes than in control cells. Further, cyclosporin that inhibits the development of mitochondrial transition pores blocked palmitate-induced alteration in mitochondrial function and palmitate-induced cell death. We further demonstrated the selectivity of cyclosporin A for the prevention of apoptotic cell death in the heart as there was no alteration in necrotic cell death produced by palmitate with cyclosporin pretreatment. Our data demonstrate the nature of palmitate-induced cell death in cardiomyocytes (both apoptotic and necrotic), propose a mitochondrial basis for its pathogenesis and show that cyclosporin A prevents palmitate-induced apoptotic cardiomyocyte cell death.     

Neptuniibacter pectenicola sp. nov. and Neptuniibacter marinus sp. nov., two novel species isolated from a Great scallop (Pecten maximus) hatchery in Norway and emended description of the genus Neptuniibacter.

Nine isolates obtained from a great scallop hatchery in Norway were characterized using a polyphasic approach. Strains were Gram-negative, aerobic and motile rods with oxidative metabolism. Phylogenetic analysis based on the sequences of 16S rRNA and rpoB genes showed that these strains formed two different groups associated with members of the genus Neptuniibacter. DNA–DNA hybridization (DDH) and Average Nucleotide Identity (ANI) demonstrated that the isolates constituted two novel species of this genus, which can be phenotypically differentiated from their closest relatives. The names Neptuniibacter marinus sp. nov. and Neptuniibacter pectenicola sp. nov are proposed, with ATR 1.1^T (=CECT 8938^T = DSM 100783^T) and LFT 1.8^T (=CECT 8936^T = DSM 100781^T) as respective type strains.     

Rapid quantification of the delta-opioid receptor selective enkephalin DPDPE in canine cerebrospinal fluid by liquid chromatography--mass spectrometry

An atmospheric pressure ionization liquid chromatographic-mass spectrometric assay was developed and validated for the determination of D-penicillamine(2,5) enkephalin (DPDPE) in cerebrospinal fluid (CSF) from dog. DPDPE and internal standard (D-Ala(2),D-Leu(5) enkephalin=DADLE) were isolated from CSF by reversed-phase C(18) solid-phase extraction with ZipTip micro-cartridges. Aliquots of extracted eluate were injected onto an Agilent Zorbax SB C(18) column (30 x 2.2 mm; 3.5 microm) at a flow-rate of 0.4 ml/min. The isocratic mobile phase of methanol-10 mM ammonium formate (pH 3) (75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadrupole mass spectrometer. DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r(2)> or =0.991) over the concentration range 1-1000 ng/ml. The efficiency of extraction recovery was greater than 97%, and the intra-assay and inter-assay precisions were within 9% relative standard deviation. DPDPE and the internal standard were stable in the injection solvent at 4 degrees C for at least 48 h. The assay was applied to the pharmacokinetic study of intrathecal DPDPE administration in the dog animal model.     

Protein synthesis in the plastid of Plasmodium falciparum.

The plastid organelle of malarial and other apicomplexan parasites contains ribosome-like particles as well as a genome dedicated largely to specifying components of a protein expression system. We have identified plastid ribosomes using hybridization studies and show that in erythrocytic stages of Plasmodium falciparum a subset of polysomes carries plastid-specified rRNAs and mRNA, supporting the idea that protein synthesis is active in the plastid.     

Antibiotic inhibitors of organellar protein synthesis in Plasmodium falciparum.

Elongation factor Tu (EF-Tu) is encoded by the tuf gene of the plastid organelle of the malaria parasite Plasmodium falciparum. A range of structurally unrelated inhibitors of this GTP-dependent translation factor was shown to have antimalarial activity in blood cultures. The most active was the cyclic thiazolyl peptide amythiamicin A with an IC50 = 0.01 microM. Demonstrable complexes were formed in vitro between a recombinant version of P. falciparum EF-Tu(pl) and inhibitors that bind to different sites on EF-Tu; these included the antibiotics kirromycin, GE2270A and enacyloxin IIa.