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.     

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.     

Analysis of N,N-diethyl-m-toluamide in porcine skin perfusates using solid-phase extraction disks and reversed-phase high-performance liquid chromatography

N,N-Diethyl-m-toluamide (DEET) is frequently used as an insect repellent by military and civilian populations. Because dermal exposure has resulted in several cases of DEET toxicosis, there is a need to rapidly and reliably determine DEET concentrations in biological matrices. An improved method for the analysis of DEET was developed for determining transdermal diffusion of low levels of DEET following application to an in vitro porcine skin flow-through diffusion cell system. The technical improvement involved the use of disk solid-phase extraction (SPE) instead of packed-bed SPE. The disk SPE method required small volumes of preconditioning, wash, and elution solvent (0.5-1 ml) to extract DEET from perfusate samples containing bovine serum albumin (BSA). The limit of quantitation (LOQ) was estimated as 0.08 micro g/ml DEET and recoveries from BSA media samples spiked with DEET ranged from 90.1 to 117% with relative standard deviation (RSD) ranging from 2.0 to 13.1%. This method was used to analyze perfusate samples from skin (n=4) topically exposed to DEET-ethanol formulations. The data from these analyses determined that DEET permeability in porcine skin was 2.55 x 10(-5)+/-0.54 x 10(-5) cm/h.     

Site-specific fluorescent derivatization and liquid chromatographic-mass spectrometric characterization of long R(3) IGF-I for bioanalytical applications

Recombinant Long R(3) IGF-I was derivatized with fluorescein isothiocyanate (FITC) at a single location by careful selection of reaction conditions (i.e. pH, and FITC/protein amino group ratio). High-performance liquid chromatography (LC) and electrospray mass spectrometry (MS) were used to confirm the extent of fluorescein conjugation. The protein conjugate was isolated and subjected to cyanogen bromide (CNBr) cleavage, followed by LC-MS to determine the site of modification. The isolated species of Long R(3) IGF-I-FITC was labeled at the N-terminal Met residue. Recognition of this fluorescent analog by monoclonal anti-IGF-I was preserved, indicating its potential for immunodiagnostic applications.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of the enantiomers of a novel anticonvulsant, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine, in rats

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the enantiomers of a novel anticonvulsant agent, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine (AAP-Cl), in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation was accomplished by an HPLC system consisting of a Chirex chiral column (250 mm x 4.6 mm i.d.) and a mobile phase of hexane:ethanol:tetrahydrofuran (280:20:40 (v/v)) containing trifluroacetic acid (0.3% (v/v)) and triethylamine (0.018% (v/v)) at a flow rate of 0.8 ml/min with UV detection. Male Sprague-Dawley rats were given (+)-AAP-Cl (10 and 20 mg/kg), (-)-AAP-Cl (10 mg/kg) or the racemic mixture (20 mg/kg) by i.v. bolus injection and serial blood samples were collected at different times after drug administration. (+)-AAP-Cl and (-)-AAP-Cl were separated with a resolution factor, Rs, of at least 1.4, and a separation factor, alpha, greater than 1.09. Linear calibration curves were obtained over the concentration range of 0.5-30 microg/ml in plasma for both (+)-AAP-Cl and (-)-AAP-Cl (R2 > or = 0.996) with a limit of quantitation of 100 ng/ml and the recovery was greater than 80% for both enantiomers. The accuracy and precision for both enantiomers ranged from 96 to 102% (+/-0.2-7%) at upper and lower concentrations. The plasma concentration-time profiles of the enantiomers of AAP-Cl were best described by a two-compartment open model with a mean terminal half-life of about 5h, volume of distribution at steady state of 3 l/kg and clearance of about 0.6l/(hkg) in rats. There was no significant difference between the pharmacokinetic parameters of (+)-AAP-Cl and (-)-AAP-Cl, suggesting that the disposition of AAP-Cl in rats is not enantioselective. In addition, no chiral inversion of (+)-AAP-Cl to (-)-AAP-Cl or vice versa was observed. The results of this investigation have shed some light on the mechanism of action and disposition of AAP-Cl in rats.     

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.