Effect of Conditioned Media from Several Cell Types on Invasion by Eimeria adenoeides Sporozoites1

ABSTRACT. The effect of conditioned media (media aspirated from a variety of cell cultures after 4 d of growth) on cellular invasion by sporozoites of the turkey coccidium, Eimeria adenoeides, was examined. Conditioned medium from turkey kidney cells and baby hamster kidney cells failed to alter invasion. However, conditioned medium from turkey cecal cell cultures produced a significant (P ≤ 0.05), two-fold increase in invasion over control medium in a variety of cell types. Retentates of conditioned medium from the turkey cecal cells that were passed through microconcentrators having molecular mass cutoffs of 50, 100, and 300 kDa similarly enhanced invasion over retentates from control medium. However, retentates from microconcentrators with a cutoff of 1,000 kDa failed to enhance invasion. Pretreatment in conditioned medium, followed by washing of sporozoites prior to inoculation into cultures, did not result in enhanced invasion. Moreover, when the interval between inoculation of sporozoites into cells and fixation of cultures was reduced to less than 3 h, no enhancement of invasion occurred. Conditioned medium from turkey cecal cells that were grown in the presence of ³⁵S-translabel had at least two labeled bands at 150 kDa and > 200 kDa that were absent in conditioned media from turkey kidney and baby hamster kidney cells.     

Pr-SNTX,a short-chain three-finger toxin from Papuan pigmy mulga snake,is an antagonist of muscle-type nicotinic acetylcholine receptor (α2βδε)

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis. Pr-SNTX protein inhibited muscle-type (α₂βδε), but not neuronal α7 nicotinic acetylcholine receptor (nAChR) activity.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

Genetic analysis of populations of threatened snake species using RAPD markers

Snakes are a particularly threatened vertebrate taxon, with distributions of many species and populations becoming increasingly fragmented. At present, little is known about the degree of genetic differentiation that exists between isolated populations even though such information may be critical to their survival and conservation. As an example of how recently developed RAPD genetic markers can be used in conservation genetics, we present preliminary results from a study which used these DNA-based markers to assess population divergence in two threatened Canadian snakes, the black rat snake ( Elaphe o. obsoleta ) and the eastern massasauga rattlesnake ( Sistrurus c. catenatus ). We present information on the levels of variation and reliability of amplification for fragments generated from five primers. We then use a recently developed analytical technique to estimate levels of nucleotide diversity within populations and sequence divergence between populations. Our results show that intrapopulation levels of divergence as estimated by the methods of Clark & Lanigan ( Molecular Biology and Evolution 1993, 10 , 1096–1111) approximate those found for mtDNA in vertebrates and that diversity between snake populations is small and non-significant when tested using randomization procedures. Thus, our study provides an example of how RAPDs can be applied to conservation genetic studies of vertebrates and suggest that the snake populations we examined have only recently become isolated and maybe considered genetically equivalent from a conservation perspective, although this conclusion needs to be confirmed with other DNA-based markers.     

HOMOGENEITY OF THE GENETIC VARIANCE-COVARIANCE MATRIX FOR ANTIPREDATOR TRAITS IN TWO NATURAL POPULATIONS OF THE GARTER SNAKE THAMNOPHIS ORDINOIDES

Quantitative genetic models of evolution rely on the genetic variance-covariance matrix to predict the phenotypic response to selection. Both prospective and retrospective studies of phenotypic evolution across generations rely on assumptions about the constancy of patterns of genetic covariance through time. In the absence of robust theoretical predictions about the stability of genetic covariances, this assumption must be tested with empirical comparisons of genetic parameters among populations and species. Genetic variance-covariance matrices were estimated for a suite of antipredator traits in two populations of the northwestern garter snake, Thamnophis ordinoides. The characters studied include color pattern and antipredator behaviors that interact to facilitate escape from predators. Significant heritabilities for all traits were detected in both populations. Genetic correlations and covariances were found among behaviors in both populations and between color pattern and behavior in one of the populations. Phenotypic means differed among populations, but pairwise comparisons revealed no heterogeneity of genetic parameters between the populations. The structure of the genetic variance-covariance matrix has apparently not changed significantly during the divergence of these two populations.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells

Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with [³⁵S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with [³H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of [³H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally.     

Entamoeba histolytica: cytopathogenicity of intact amebae and cell-free extracts; isolation and characterization of an intracellular toxin.

Toxicity assays on cell-free extracts of virulent and nonvirulent strains of Entamoeba histolytica were carried out in microtiter plates. These extracts had a cytopathogenic effect (CPE) on monolayers of baby hamster kidney cells. CPE was inhibited by normal human serum, fetal calf serum, and other sera, probably due to their IgG component. Using gel chromatography the toxic material, a protein, was found in a fraction with molecular weight between 35,000 and 45,000. This fraction contained a strong glycoprotein antigen. CPE caused by this toxin differs in several ways from the earlier described “contact lysis” caused by intact amebae. The possible significance of these two modes of toxicity of Entamoeba histolytica for the pathogenesis of amebiasis is discussed.