Amblyomma americanum: characterization of salivary prostaglandins E2 and F2 alpha by RP-HPLC/bioassay and gas chromatography-mass spectrometry.

Secretagogue-induced saliva of the tick, Amblyomma americanum (L.) was fractionated by reversed-phase-high-performance liquid chromatography (RP-HPLC) and bioassayed in smooth muscle preparations. Material with retention times of authentic prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) were found to cause contraction of preparations of rat colon and rat stomach strips. Gas chromatography-mass spectra of selected ions of both HPLC-purified fractions confirmed the existence of PGE2 and PGF2 alpha. Bioassay of individual samples obtained from ticks stimulated to salivate with pilocarpine, dopamine + theophiline, or dopamine + theophiline + GABA indicated that all these secretagogues induced similar amounts of prostaglandin secretion, averaging 469 ng PGE2/ml. These pharmacological doses of prostaglandin are hypothesized to assist in tick feeding by inducing vasodilation and/or other pharmacological events in their hosts.     

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.     

A molecular thermodynamic approach to predict the secondary structure of homopolypeptides in aqueous systems.

Under physiological conditions, many polypeptide chains spontaneously fold into discrete and tightly packed three-dimensional structures. The folded polypeptide chain conformation is believed to represent a minimum Gibbs energy of the system, governed by the weak interactions that operate between the amino acid residues and between the residues and the solvent. A semiempirical molecular thermodynamic model is proposed to represent the Gibbs energy of folding of aqueous homopolypeptide systems. The model takes into consideration both the entropy contribution and the enthalpy contribution of folding homopolypeptide chains in aqueous solutions. The entropy contribution is derived from the Flory-Huggins expression for the entropy of mixing. It accounts for the entropy loss in folding a random-coiled polypeptide chain into a specific polypeptide conformation. The enthalpy contribution is derived from a molecular segment-based Non-Random Two Liquid (NRTL) local composition model [H. Renon and J. M. Prausnitz (1968) AIChE J., Vol. 14, pp. 135-142; C.-C. Chen and L. B. Evans (1986) AIChE J., Vol. 32, pp. 444-454], which takes into consideration of the residue-residue, residue-solvent, and solvent-solvent binary physical interactions along with the local compositions of amino acid residues in aqueous homopolypeptides. The UNIFAC group contribution method [A. Fredenslund, R. L. Jones, and J. M. Prausnitz (1975) AIChE J., 21, 1086-1099; A. Fredenslund, J. Gmehling, and P. Rasmussen (1977) Vapor-Liquid Equilibrium Using UNIFAC, Elsevier Scientific Publishing Company, Amsterdam], developed originally to estimate the excess Gibbs energy of solutions of small molecules, was used to estimate the NRTL binary interaction parameters. The model yields a hydrophobicity scale for the 20 amino acid side chains, which compares favorably with established scales [Y. Nozaki and C. Tanford (1971) Journal of Biological Chemistry, Vol. 46, pp. 2211-2217; E. B. Leodidis and T. A. Hatton (1990) Journal of Physical Chemistry, Vol. 94, pp. 6411-6420]. In addition, the model generates qualitatively correct thermodynamic constants and it accurately predicts thermodynamically favorable folding of a number of aqueous homopolypeptides from random-coiled states into alpha-helices. The model further facilitates estimation of the Zimm-Bragg helix growth parameter s and the nucleation parameter sigma for amino acid residues [B. H. Zimm and J. K. Bragg (1959) Journal of Chemical Physics, Vol. 31, pp. 526-535]. The calculated values of the two parameters fall into the ranges suggested by Zimm and Bragg.     

The functional morphology of the respiratory system of the Holothyrida (= Tetrastigmata) Acari: Anactinotrichida

The respiratory system of Holothyrus coccinella Gervais (Holothyridae) and Allothyrus australasiae (Womersley) (Allothyridae) were examined. The stigma-peritreme complex is connected to tracheae and ventilated by indirect muscles. The peritreme provides an alternative route for the entry of air into the tracheal system, should a stigma be occluded by debris and retards water vapour transpiration, the mechanisms of which are compared in the two species.     

The effect of inhibition of hexosemonophosphate shunt on the metabolism of glucose and function in rat brain in vivo

Rats treated 4 hr previously with 6-aminonicotinamide showed a twenty-four fold increase of [¹⁴C]phosphogluconate in the adult brain at 30 min after injection of [U-¹⁴C]glucose indicating a blockade of the hexosemonophosphate shunt. There was a significant increase in the¹⁴C-content of glucose and glucose 6-phosphate, and a decrease in that of amino acids. [¹⁴C]Phosphoglycerate content showed no consistent change after 6-aminonicotinamide treatment. The concentration of glucose and glucose 6-phosphate increased significantly without a significant change in the lactate pool in the brain of 6-aminonicotinamide treated rats. The rate of utilization of glucose in the brain of control rats was 0.73 mol/min per g of brain. It decreased by 16% in rats treated with 6-aminonicotinamide; the results suggested that both glycolysis and pyruvate oxidation were affected. The amount of glucose utilized in the brain by the hexosemonophosphate shunt was approximately 0.0093 mol/min per g of brain, i.e. 1.3% of the total rate of utilization of glucose. The observed changes were not due to hypothermia. The rate of glucose utilization was higher in animals exposed to higher ambient temperature and to stress caused by handling. The results were explained by postulating a role for the hexosemonophosphate shunt in providing neurotransmitter amino acids glutamate and -aminobutyrate, and interdependence of brain function and glucose utilization.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Spectral evidence for a component involved in hydrogen metabolism of soybean nodule bacteroids

A component with a difference spectrum similar to that of b-type cytochromes which becomes reduced upon the addition of H₂ has been demonstrated in soybean nodule bacteroids. This electron carrier, referred to as component 559-H₂, is present in hydrogenase-positive strains of Rhizobium japonicum but has not been detected in mutants that lack hydrogenase activity or in hydrogenase-negative wild-type strains. A positive correlation between concentrations of component 559-H₂ and hydrogenase activities has been established. These results provide further evidence that component 559-H₂ is involved in H₂ metabolism in R. japonicum.     

Promotion of growth and hydrogen ion efflux by auxin in roots of maize pretreated with ethylene biosynthesis inhibitors

Low concentrations of auxin (e.g. 10⁻¹⁰m) do not promote the growth of intact seedling roots of maize (Zea mays L. Bear Hybrid WF 9 × 38). Higher concentrations are inhibitory. When the roots are pretreated with the ethylene biosynthesis inhibitors, cobalt and aminoethoxyvinylglycine, auxin (10⁻¹⁰ to 10⁻⁸m) strongly promotes their growth. The promotion of growth by auxin in pretreated roots is preceded by enhanced hydrogen ion secretion from the roots. The data indicate that hormone-enhanced hydrogen ion secretion may play a role in the rapid promotion of root growth by auxin. The ability of auxin to promote the growth of intact roots is discussed in relation to the Cholodny/Went hypothesis of hormonal control of root geotropism.     

Extrahelical bases in duplex DNA

The two non-complementary synthetic DNAs, poly[d(G-G-A)] and poly[d(T-C)] can form a duplex structure at moderate ionic strengths in which every other T is extrahelical:

Only this structure is consistent with the stoichiometry of formation and with the production of C? photodimers on ultraviolet light irradiation. Further characterization with respect to melting temperature and interaction with nuclease or damage-recognizing proteins is described.