AGGREGATION AND TRANSFORMATION OF RAT LYMPHOCYTES ON RAT EMBRYO MONOLAYERS

Lymphocytes from Sprague-Dawley rats have been cultured on monolayers of embryo-derived fibroblasts from the same outbred strain. Under these conditions the lymphocytes form aggregates, and transformation of small lymphocytes to lymphoid blast cells occurs within these aggregates. Transformation is characterized cytologically by enlargement of the nucleus, dispersion of nuclear chromatin, and the appearance of a prominent nucleolus. The principal cytoplasmic changes are an increase in cytoplasmic volume, a marked increase in number of ribosomes, and a clustering of ribosomes. These changes parallel those seen in the transformation of lymphocytes caused by a variety of treatments. One apparent difference is the paucity of lysosomes and lipid inclusions in the lymphocytes that transform on the monolayer.     

Studies on the antimalarial mode of action of quinoline-containing drugs: time-dependence and irreversibility of drug action, and interactions with compounds that alter the function of the parasite's food vacuole

The quinoline-containing antimalarial drugs chloroquine, quinine and mefloquine exert an irreversible inhibitory effect on erythrocytic stages of Plasmodium falciparum grown in culture. Inhibition is time- and concentration-dependent and the full effect is observed after 2-6 hours of exposure to the drug. Washing of infected cells after drug exposure in the presence of NH4Cl to accelerate drug efflux, intensifies the inhibitory effect of chloroquine, probably due to the pH-dependent release of highly concentrated drug from the acidic food vacuole of the parasite. When both antimalarials and NH4Cl are present in the culture, drug effect is reduced, as expected from the demonstrable alkalinization of the food vacuole and the consequent reduction in drug accumulation. The protease inhibitor leupeptin inhibits digestion of ingested host cell cytosol, and thus inhibits parasite growth, though reversibly so (Rosenthal et al, J. Clin. Invest. 82 1560-1566 (1988)). Thus, although the antimalarials also inhibit the feeding process, this is not the cause of their irreversible action. Leupeptin is found to be antagonistic to antimalarials' action, suggesting that the drugs form complexes with products of host cell digestion that are responsible for irreversible inhibition of parasite growth.     

Reassembled plasma low density lipoproteins. Phospholipid-cholesterol ester-apoprotein B complexes

Reassembled low density lipoprotein (LDL) complexes have been prepared by the interaction of lipid-free sodium deoxycholate-solubilized apoprotein B (apoB) of native human LDL with preformed, 200 A in diameter, microemulsions of cholesteryl oleate (CO), surface-stabilized by either egg yolk phosphatidylcholine ( EYPC ) or dimyristoyl phosphatidylcholine (DMPC). Gel chromatography of PC/CO/apoB complexes shows co-elution of the complex at 43% PC, 43% CO, and 14% apoB. Negative stain electron microscopy shows the particles to be circular, homogeneous, and approximately 200 A in diameter. PC/CO/apoB complexes exhibit beta-migration on agarose gels and show one high molecular weight protein band on 3.0% sodium dodecyl sulfate-polyacrylamide gels. Differential scanning calorimetry and x-ray scattering show the lipids in the complexes to undergo at least two specific thermal transitions depending on lipid composition, one associated with the core-located cholesterol esters similar to LDL and the protein-free microemulsions and the other from the phospholipid forming the surface monolayer. In addition, particle disruption-protein unfolding/denaturation occur irreversibly at 80-85 degrees C. At 4 degrees C, the secondary structure of apoB on complexes of EYPC /CO/apoB is similar to that of native LDL. For complexes of DMPC/CO/apoB, the secondary structure shows less alpha-helix which correlates with the difference in surface lipid environment. The reassembled complexes of PC/CO/apoB provide a defined system in which the components may be varied systematically in order to study the molecular organization, molecular interactions, and metabolism of LDL.     

Mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase

The release of Zn2+ from aspartate transcarbamoylase (ATCase; c6r6) upon challenge by p-hydroxymercuriphenylsulfonate (PMPS) has been studied using the sensitive, high-affinity metallochromic indicator 4-(2-pyridylazo)resorcinol at pH 7.0. When the--SH group of each catalytic (c) chain is protected, 1 Zn2+ is released for every 4 eq of PMPS added to ATCase during titration of the 24--SH groups of regulatory (r) chains. Moreover, the release of Zn2+ is a linear function of PMPS added, indicating that the rate-limiting step in Zn2+ release is mercurial attack on the 1st of the 4 r--SH groups bonded tetrahedrally to Zn2+ in an r chain near c:r contacts. Dissociation of ATCase is linked to Zn2+ release and mercaptide formation; e.g. upon addition of 4 eq of PMPS to ATCase in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, 1/6th of ATCase is dissociated to c3 and r2 subunits at approximately 83% of the rate of Zn2+ release, with no accumulation of the c6r4 intermediate as is observed in KPO4 buffer. Adding less than or equal to 4 PMPS/ATCase, the release of Zn2+ is first-order in [PMPS] and is virtually independent of [ATCase] with an activation energy of 18 kcal/mol. With large excesses of PMPS, stopped-flow traces show a lag period followed by pseudo first-order release of Zn2+ from ATCase and the reaction order in [PMPS] = approximately 1.3. Under these conditions, PMPS has a chaotropic effect on ATCase; the activation energy for Zn2+ release is much lower than that obtained with limiting PMPS and is increased by the presence of phosphate or active-site ligand from 6.6 to approximately 12 kcal/mol. A reasonable explanation of the observed kinetic data is that the organomercurial reagent binds reversibly to nitrogenous side chain groups in an ATCase molecule prior to the rate-limiting reaction with a sulfhydryl group.     

Monoclonal antibodies PMN 6, PMN 29, and PM-81 bind differently to glycolipids containing a sugar sequence occurring in lacto-N-fucopentaose III

Three monoclonal antibodies, PMN 6, PMN 29, and PM-81, bind myeloid cells. Antibodies PMN 6 and PMN 29 bind specifically to granulocytes but differ in their ability to bind some other cell lines [E. D. Ball, R. F. Graziano, L. Shen, and M. W. Fanger (1982) Proc. Natl. Acad. Sci. USA 79, 5374-5378]. Antibody PM-81, in addition to granulocytes, also binds to eosinophils, monocytes, and most acute myelocytic leukemia cells [E. D. Ball, R. F. Graziano, and M. W. Fanger (1983) J. Immunol. 130, 2937-2941]. Despite these differences, the binding of all three antibodies to cells was inhibited by the oligosaccharide, lacto-N-fucopentaose III [Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc]. Solid-phase radioimmunoassays using purified glycolipids containing sugar sequences found in lacto-N-fucopentaose III demonstrated different binding characteristics for each antibody. PM-81 bound lower concentrations of glycolipids than PMN 29, while PMN 6 required the highest concentration of glycolipids for binding. Autoradiography of thin-layer chromatograms of glycolipid antigens supported these results. The binding of these monoclonal antibodies to cells probably depends on the density of antigens on the cell surface, each antibody requiring a different density. Thus, cells containing antigen below a certain threshold concentration may not bind low-affinity antibodies.     

On the possibility of pleiotropic monogenic control of hereditary polyposis and primary cancer of the colon.

The results of the segregation analysis of hereditary adenomatous polyposis and primary cancer of the colon are given, taking into account the age-dependent manifestation of genotypes and the specificity of pedigree sampling. The results are used for testing the hypothesis of pleiotropic monogenic control of these two colon pathologies.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Primordial germ cells in the mouse embryo during gastrulation

With the aid of a whole-mount technique, we have detected a small cluster of alkaline phosphatase (ALP)-positive cells in whole mounts of mid-primitive-streak-stage embryos, 7-7 1/4 days post coitum (dpc). Within the cluster, about 8 cells contain a small cytoplasmic spot, intensely stained for ALP activity and possibly associated with an active Golgi complex. The cluster lies just posterior to the definitive primitive streak in the extraembryonic mesoderm, separated from the embryo by the amniotic fold. Towards the end of gastrulation, the number of cells containing the ALP-positive spot rises to between 50 and 80. Thereafter the number of cells in the extraembryonic cluster declines, and similar cells start to be seen in the mesoderm of the primitive streak and then in the endoderm. At 8 dpc, about 125 ALP-stained cells are found, mainly in the hindgut endoderm and also at the base of the allantois, their appearance and location at this stage agreeing closely with previous reports on primordial germ cells (PGCs). Embryos from which the cluster area has been removed at the 7-day stage are devoid of PGCs after culture for 48 h, whereas the excised tissue is rich in PGCs. We argue that the cells in the cluster are indeed primordial germ cells, at a stage significantly earlier than any reported previously. This would indicate that the PGC lineage in the mouse is set aside at least as early as 7 dpc, possibly as one of the first 'mesodermal' cell types to emerge, and that its differentiation, as expressed by ALP activity, is gradual.     

Metabolic interconnection between the human malarial parasite Plasmodium falciparum and its host erythrocyte. Regulation of ATP levels by means of an adenylate translocator and adenylate kinase

The metabolic inter-relationships between malarial parasites and their host erythrocytes are poorly understood. They have been investigated hitherto mostly by observing parasite behavior in erythrocyte variants, in metabolically altered erythrocytes, or in cell-free in vitro systems. We have studied the interconnection between the bioenergetic metabolism of host and parasite through compartment analysis of ATP in Plasmodium falciparum-infected human red blood cells, using Sendai virus-induced host cell lysis. ATP concentrations in host and parasite compartments were found to be equal. Inhibitors of mitochondrial activity reduce ATP levels to a similar extent in host and parasite compartments, although only the parasite contains functional mitochondria. It is shown that equalization of ATP levels is brought about by means of an adenylate translocator, probably localized at the parasite plasma membrane, in conjunction with adenylate kinase activity detected both in host and parasite compartments. The translocator is inhibited by compounds which are known to inhibit specifically the translocator of the inner membrane of mammalian mitochondria, with identical inhibitory constants. Addition of these inhibitors to intact infected cells causes a rapid depletion of ATP in the host compartment and a parallel increase in the parasite, suggesting that the parasite supplies ATP to its host cell rather than the reverse.     

Antistasin, an inhibitor of coagulation and metastasis, binds to sulfatide (Gal(3-SO4) beta 1-1Cer) and has a sequence homology with other proteins that bind sulfated glycoconjugates

Antistasin, a 15-kDa salivary protein from the Mexican leech Haementeria officinalis, inhibits both blood coagulation and the metastasis of tumors (Tuszynski, G. P., Gasic, T. B., and Gasic, G. J. (1987) J. Biol. Chem. 262, 9718-9723). Antistasin binds to heparin-agarose, suggesting the protein interacts with sulfated glycoconjugates. The specificity of the interaction between antistasin and heparin was tested by measuring the binding of antistasin to various lipids and by comparing the ability of several charged glycoconjugates to inhibit binding. Of the lipids tested, antistasin binds with high affinity only to sulfatide (Gal(3-SO4)beta 1-1Cer) and does not bind to comparable levels of phospholipids, neutral glycosphingolipids, gangliosides, or cholesterol-3-SO4. The binding of antistasin to sulfatide is inhibited by dextran sulfate, fucoidan, and heparin, with I50 values of 1.5, 9.2, and 16 micrograms/ml, respectively. Comparable levels of chondroitin sulfates A, B, C, keratan sulfate, or hyaluronic acid do not inhibit binding. Comparisons of the amino acid sequences of antistasin and other sulfatide or heparin-binding proteins revealed a region of homology, based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg, which may be a sulfated glycoconjugate binding domain. In addition, homologies were found with the alternate complement pathway protein properdin and coat proteins from malaria circumsporozoites and Herpes simplex I.