Culture and properties of cells derived from Kaposi sarcoma

We describe the establishment of four continuous cell cultures isolated from pleural or peritoneal fluid of patients with Kaposi sarcoma (KS) and show evidence that these cells are derived from vascular endothelium. Although provision of an extracellular matrix (fibronectin, laminin, or matrigel) was essential, the cell cultures were not dependent on exogenously added growth factors (platelet-derived growth factor, epidermal growth factor with or without heparin) for continuous culture. Specific staining for endothelial cell (EC) markers (factor VIII, Ulex europaeus type 1 lectin) and the secretion of endothelin, a vascular EC product, were demonstrated. The KS cells secreted large amounts of cytokines (granulocyte-macrophage-CSF, TNF-alpha, IL-1 beta, and especially IL-6). Conditioned media from the KS cells caused normal capillary EC to proliferate. The KS cells synthesized fibroblast growth activity in amounts sufficient to induce the proliferation of normal EC and fibroblasts. These data support the existence of a paracrine pathway of EC proliferation in KS and suggest that KS cells could sustain their own growth via an autocrine mechanism.     

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.     

Molecular modeling of proteins: a strategy for energy minimization by molecular mechanics in the AMBER force field.

Energy minimization is an important step in molecular modeling of proteins. In this study, we sought to develop a minimization strategy which would give the best final structures with the shortest computer time in the AMBER force field. In the all-atom model, we performed energy minimization of the melittin (mostly alpha-helical) and cardiotoxin (mostly beta-sheet and beta-turns) crystal structures by both constrained and unconstrained pathways. In the constrained path, which has been recommended in the energy minimization of proteins, hydrogens were relaxed first, followed by the side chains of amino acid residues, and finally the whole molecule. Despite the logic of this approach, however, the structures minimized by the unconstrained path fit the experimental structures better than those minimized by constrained paths. Moreover, the unconstrained path saved considerable computer time. We also compared the effects of the steepest descents and conjugate gradients algorithms in energy minimization. Previously, steepest descents has been used in the initial stages of minimization and conjugate gradients in the final stages of minimization. We therefore studied the effect on the final structure of performing an initial minimization by steepest descents. The structures minimized by conjugate gradients alone resembled the structures minimized initially by the steepest descents and subsequently by the conjugate gradients algorithms. Thus an initial minimization using steepest descents is wasteful and unnecessary, especially when starting from the crystal structure. Based on these results, we propose the use of an unconstrained path and conjugate gradients for energy minimization of proteins. This procedure results in low energy structures closer to the experimental structures, and saves about 70-80% of computer time. This procedure was applied in building models of lysozyme mutants. The crystal structure of native T4 lysozyme was mutated to three different mutants and the structures were minimized. The minimized structures closely fit the crystal structures of the respective mutants (less than 0.3 A root-mean-square, RMS, deviation in the position of all heavy atoms). These results confirm the efficiency of the proposed minimization strategy in modeling closely related homologs. To determine the reliability of the united atom approximation, we also performed all of the above minimizations with united atom models. This approximation gave structures with similar but slightly higher RMS deviations than the all-atom model, but gave further savings of 60-70% in computer time. However, we feel further investigation is essential to determine the reliability of this approximation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of v-src induces aberrant development and twinning in chimaeric mice

The role of the proto-oncogene c-src in mouse development has been investigated by studying the consequences of expressing its viral homologue, v-src. Embryonic stem (ES) cell lines with differing levels of v-src tyrosine kinase activity have been used to generate chimaeric mice. Whereas a low level of v-src expression is compatible with embryogenesis, chimaeras derived from ES clones with high levels of v-src activity develop abnormally and die on the 8th-9th day of gestation. These abnormalities are characterized by the formation of twin or multiple embryos within the same Reichert's membrane, and by the arrest of embryonic development at the late egg cylinder stage, accompanied by relative expansion of the visceral yolk sac (VYS) and hyperplasia of the VYS endoderm. These results demonstrate for the first time that deregulated expression of the src protooncogene product can induce developmental abnormalities during early embryogenesis.