Chemoattraction of a bactivorous ciliate to bacteria surface compounds

The locomotory response to cell surface compounds extracted from two prey species,Vibrio natriegens andVibrio neries, was tested for a bacterivorous ciliate,Pseudochnilembus marinus Thompson 1966. Chemoattraction of the ciliate to the surface compounds stabilized in agarose baits was not equal for the two prey species. Fractionation of the extracts suggested the attractive substance was a high molecular weight compound. The expression of the differential response was dependant on the physiological condition and prior prey species exposure of the ciliate test population. The recognition and response to material normally found on the surface of prey cells supports evidence for the involvement of chemical sensing of gradients of prey particles and dissolved compounds of prey origin in the natural swimming behavior of bacterivorous ciliates. The prey species-specific reactions and influence of ciliate physiological state on chemosensory response suggest ciliate-bacteria interactions may be more complex than preciously assumed.     

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.     

Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein and not homologous CEA-related glycoproteins of other species. These results suggest that MHV-A59 binds to a mouse-specific epitope of MHVR, and they support the hypothesis that the species specificity of MHV-A59 infection may be due to the specificity of the virus-receptor interaction.     

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.     

Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding.

The Escherichia coli D-galactose and D-glucose receptor, an aqueous periplasmic receptor that triggers sugar sensing and transport, possesses a single Ca2+ binding site similar in structure and specificity to the EF-hand class of sites found in eukaryotic Ca2+ signaling proteins including calmodulin and its homologues. A universal feature of these sites is the use of a pentagonal bipyramidal array of seven oxygens to coordinate bound Ca2+. Here we investigate the mechanisms used by this coordinating array to control ion specificity. To vary the cavity size and charge of the array, we have replaced axial glutamine 142 in the prokaryotic site with asparagine, glutamate, and aspartate. The ion selectivities of the resulting engineered sites have been quantitated by measuring dissociation constants for a series of spherical metal ions, differing in increments of radius and charge, from groups Ia, IIa, and IIIa and the lanthanides. Dramatic specificity changes are observed: sites containing an engineered smaller side chain (Asn or Asp) bind the largest cations up to 50-fold more tightly than the native site; and sites containing an engineered negative side chain (Glu or Asp) exhibit preferences for trivalent over divalent cations up to 1900-fold higher than the native site. The results indicate that the cavity size and negative charge of the coordination array play key roles in selective Ca2+ binding and that the array can be engineered to preferentially bind other cations.     

Sequence alignment of the G-protein coupled receptor superfamily.

The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.     

Development of specific functionally active receptors for platelet-activating factor in HL-60 cells following granulocytic differentiation

A human promyelocytic leukemia cell line (undifferentiated HL-60 cells) as well as a granulocyte form of HL-60 cells induced in vitro by exposure to dimethyl sulfoxide were examined for binding, metabolism, and biological responses to platelet-activating factor (PAF). Undifferentiated and differentiated HL-60 cells each exhibit a high capacity to incorporate and metabolize [3H]PAF at 37 degrees C; however, the amount of [3H]PAF that is assimilated by both cell populations is greatly reduced and its metabolism abolished at less than or equal to 4 degrees C. At 0 degrees C HL-60 granulocytes bind more [3H]PAF than their undifferentiated counterparts. Binding to differentiated cells reaches equilibrium within 80 min and is saturable, reversible and specific; PAF receptor antagonists WEB 2086, L-659,989, BN 52021, and kadsurenone abolish this specific [3H]PAF binding. In contrast, [3H]PAF uptake by undifferentiated HL-60 cells is neither saturable nor sensitive to specific receptor antagonists. Scatchard analyses reveal 5850 +/- 850 binding sites per differentiated HL-60 cell with a dissociation constant of 0.66 +/- 0.15 nM. In the presence of cytochalasin B, PAF (200 nM) induces degranulation only in differentiated cells and this response also is blocked by PAF receptor antagonists. Our results demonstrate that HL-60 cells develop specific and functionally active PAF receptors only after chemically induced differentiation into granulocytes.     

L-carnitine delays the killing of cultured hepatocytes by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The role of fatty acid metabolism in chemical-dependent cell injury is poorly understood. Addition of L-carnitine to the incubation medium of cultured hepatocytes delayed cell killing initiated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Protection by L-carnitine was stereospecific and observed as late as 1 h following addition of MPTP. D-Carnitine, but not iodoacetate, reversed the L-carnitine effect. Monoamine oxidase A and B activities, MPTP/N-methyl-4-phenyl-pyridinium levels, and MPTP-dependent loss of mitochondrial membrane potential measured by release of [3H]triphenylmethylphosphonium were not altered by addition of L-carnitine. Significant changes in MPTP-induced depletion of total cellular ATP did not occur with excess L-carnitine. Although the mechanism of cytoprotection exerted by L-carnitine remains unresolved, the data suggest that L-carnitine does not significantly alter: (i) mitochondrial-dependent bioactivation of MPTP; (ii) MPTP-dependent loss of mitochondrial membrane potential; or (iii) MPTP-mediated depletion of total cellular ATP content. We conclude that alterations of fatty acid metabolism may contribute to the toxic consequences of exposure to MPTP. Moreover, the lack of L-carnitine-mediated cytoprotection of monolayers incubated with 4-phenylpyridine or potassium cyanide suggests: (i) a link between fatty acid metabolism and mitochondrial membrane-mediated, bioactivation-dependent cell killing; and (ii) that inhibition of NADH dehydrogenase may not totally explain the mechanism of MPTP cytotoxicity.     

Nucleoside Diphosphate Levels in Taproots and Fibrous Roots of Beta vulgaris L. : Implifications for Control of Sucrose Allocation between Two Sinks

Sucrose synthase (EC 2.4.1.13) of sugarbeet (Beta vulgaris L.) fibrous roots has a distinctly different order of preference for nucleoside diphosphate substrates than that of the taproot enzyme (Silvius, Snyder 1979 Plant Physiol 64: 1070-1073). Separation and quantitation of UDP, ADP, and GDP in root extracts by high-pressure liquid chromatography revealed that UDP levels in taproot tissue were 5 to 10 times greater than those of fibrous root tissues. The lower fibrous root UDP levels were associated with significantly higher ADP and GDP levels in these roots as compared to the taproot. These differences are consistent with differences in the substrate affinity of sucrose synthase and suggest a regulatory role of the enzyme in the control of sucrose cleavage and utilization between the two root types.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.