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.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

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.     

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.     

Effects of 9-cis- and all-trans-retinoic acids on blood glucose homeostasis in the fiddler crab, Uca pugilator

9-cis-Retinoic acid (9CRA) and all-trans-retinoic acid (ATRA) are known to be involved in the regulation of glucose homeostasis in vertebrates by inducing insulin release and expression of glucose transporter proteins. In view of the fact that both 9CRA and ATRA are endogenous to the fiddler crab, Uca pugilator, that a retinoid X receptor exists in this fiddler crab and that activities of insulin-like and insulin-like growth factor-like peptides have been reported for crustaceans, we investigated whether 9CRA and ATRA also play a role in glucose homeostasis in U. pugilator. Neither 9CRA nor ATRA was found to produce hypoglycemic effects at a dose of 10 microg/g live mass. However, 9CRA, but not ATRA, induced hyperglycemia. Such 9CRA-induced hyperglycemia was apparently mediated by the eyestalk hormone CHH since injection of 9CRA into eyestalk-ablated crabs did not result in hyperglycemia. ATRA was found to have an inhibitory effect on the recovery of blood glucose concentration following ATRA administration. Discussion on the possible mechanisms for the actions of 9CRA and ATRA was presented.     

Phenetic analysis of the Mexican midland pinyon pines, Pinus cembroides and Pinus johannis

This account presents an analysis of allopatric, sympatric and juxtaposed populations of the pinyon pines, Pinus cembroides Zucc. and P. johannis M.-F. Robert, based on phenetic analysis of morphological data, and draws on a comparison and differentiation of P. culminicola Andresen & Beaman. A qualitative and quantitative database for populations has been assembled and used for a cluster and ordination analysis using NTSYS. It was found that individual populations of the taxa were distinguishable. The emergence of morphological intermediate populations between P. cembroides and P. johannis was confirmed. The pattern of variation in P. johannis was found to be discrete, showing three geographical groups. The phenetic similarity between P. johannis from Zacatecas and P. johannis from San Luis Potosi and Queretaro was lower than that of P. johannis from Zacatecas and P. culminicola from Nuevo Leon. The following traits distinguish P. cembroides and the other two species: colour and fertility of seeds; cone shape; colour and width of needles; physiognomy; seed dimensions. This last trait can be used to distinguish three subgroups of pines with white endosperm in the northern, central and southern regions of the Mexican highlands.     

Phylogenetic analysis of the SSU rRNA from members of the Chrysophyceae

The nucleotide sequence for the nuclear-encoded small subunit ribosomal RNA gene (SSU rRNA) was determined for 24 species of the Chrysophyceae sensu stricto. These sequences were aligned, using primary and secondary structure, with nine previously published sequences for the Chrysophyceae, 14 for the Synurophyceae, and five for the Eustigmatophyceae (outgroup). Data analyses were the substitution rate calibration distance method using neighbor-joining (TREECON), Kimura 2-parameter neighbor-joining method (PAUP) and the maximum parsimony method (PAUP, PHYLIP). Trees from the analyses were largely congruent, but bootstrap support was weak at many nodes. The analyses recovered clades of uniflagellate and biflagellate organisms associated with current higher level taxonomy (e.g., subclass, order). The genus Ochromonas was polyphyletic, and O. tuberculata in particular was distantly related to the other Ochromonas species in the analysis. The family Paraphysomonadaceae occupied a basal position in three of four analyses. The class Synurophyceae appeared to be embedded within the Chrysophyceae, but bootstrap support was weak (< 50%) in all analyses except the PHYLIP parsimony analysis (= 81%). It was considered premature to place the Synurophyceae back into the Chrysophyceae based upon the analysis of one gene, especially given the ultrastructural and pigment differences between the two groups, but the relationship of these two groups deserves further study.     

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.