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adherence of acanthamoeba castellanii to new daily wear, extended wear, and disposable soft contact lenses.we studied the adherence of acanthamoeba to three types of new, unworn soft contact lenses: daily wear; extended wear; and disposable contact lenses. we used a human corneal isolate of acanthamoeba castellanii in commercially prepared saline. unworn lenses were exposed to acanthamoeba cysts or trophozoites with stirring. after exposure the numbers of acanthamoeba adhering were determined by light microscopy and confirmed by scanning electron microscopy. in general, the adherence of acanthamoeba ...19912049818
ultraviolet radiation for the sterilization of contact lenses.two sources of ultraviolet (uv) radiation with peak wavelengths in the uv-c or uv-b ranges were compared for their ability to sterilize contact lenses infected with pseudomonas aeruginosa, streptococcus pneumoniae, acanthamoeba castellani, candida albicans, and aspergillus niger. also examined was the effect of prolonged uv light exposure on soft and rigid gas permeable (rgp) contact lenses. the uv-c lamp (253.7 nm, 250 mw/cm2 at 1 cm) was germicidal for all organisms within 20 minutes but cause ...19902123422
phagocytosis in acanthamoeba: i. a mannose receptor is responsible for the binding and phagocytosis of yeast.we have examined the initial events in phagocytosis by acanthamoeba castellanii in order to understand this process at the molecular level and have determined that phagocytosis in this organism is mediated by a receptor which recognizes mannose-rich elements in the particle to be phagocytosed. we demonstrate that the binding and internalization of yeast particles can be inhibited by the sugars (d(+)-mannose and d(-)-fructose in a stereospecific, concentration-dependent manner. this inhibition is ...19902125603
characterization of potentially pathogenic free-living amoebae in sewage samples of calcutta, india.1. it is widely accepted that foul or polluted environments are the principal sources of potentially pathogenic species of free-living amoebae. the present paper is the first report of occurrence of potentially pathogenic free-living amoebae in sewage samples of calcutta, india. 2. we describe the occurrence, isolation, specific identification and comparative mouse pathogenicity test of two pathogenic amoebae, viz., naegleria fowleri (n. aerobia) carter, 1970, causing human meningoencephalitis a ...19902136561
regulation of the actin-activated atpase activity of acanthamoeba myosin ii by copolymerization with phosphorylated and dephosphorylated peptides derived from the carboxyl-terminal end of the heavy chain.myosin ii from acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. the amino-terminal approximately 90 kda of each heavy chain form a globular head that contains the atpase site and an atp-sensitive actin-binding site. the carboxyl-terminal approximately 80 kda of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. phospho ...19902141027
acanthamoeba myosin i heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation.the actin-activated mg2(+)-atpase activities of myosins i from acanthamoeba castellanii are fully expressed only when a single amino acid on their heavy chain is phosphorylated by myosin i heavy chain kinase. here we show that kinase isolated by a procedure designed to minimize its phosphorylation during purification can incorporate up to 7.5 mol of phosphate/mol of enzyme when incubated with atp, possibly by autophosphorylation. the rate of phosphorylation is enhanced about 20-fold by phosphati ...19902154483
functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of acanthamoeba myosin ii.the actin-activated mg2(+)-atpase activity of myosin ii from acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, cooh-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the cooh terminus. to investigate the essential requirements for regulation, myosin ii filaments in the presence of f-actin were digested by arginine-specific submaxillary gland protease. two-dimensional peptide mapping of pu ...19902160267
in vivo and in vitro collagenolytic activity of acanthamoeba castellanii.axenic cultures of acanthamoeba castellanii contained a collagenolytic enzyme that digested collagen shields and purified collagen in vitro. specificity of biologic activity was determined by the addition of selected enzyme inhibitors to the assays and revealed that the parasite-conditioned medium contained both collagenase and lower concentrations of other proteolytic enzymes. however, most of the collagenolytic and pathogenic activity was directly attributable to specific collagenase. intrastr ...19902173683
phagocytosis in acanthamoeba: ii. soluble and insoluble mannose-rich ligands stimulate phosphoinositide metabolism.the generation of second messengers during phagocytosis of yeast by acanthamoeba castellanii was examined. the kinetics of binding and internalization of yeast by acanthamoeba were measured and this was compared with the generation of known second messengers. we observed stimulated degradation of pi-4, 5-p2 to 1,4,5 ip3 with kinetics similar to that observed for the binding of yeast to amoeba. similar production of ip3 could be induced upon treatment with a soluble mannosylated glycoprotein. we ...19902177061
acanthamoeba keratitis and contact lens wear: the patient is at fault.the avoidance of nonsterile solutions is important to curtailing acanthamoeba keratitis, a serious infection that has been found to occur with all types of contact lens (cl) wear. increased patient education, revised recommendations regarding the use of tap and distilled water, and improved disinfecting systems are vital to preventing infection. these precautions are particularly important since it appears that acanthamoeba, unlike pseudomonas, may not require an epithelial defect to establish c ...19902189677
cdna sequence analysis of a 29-kda cysteine-rich surface antigen of pathogenic entamoeba histolytica.a gamma gt11 cdna library was constructed from poly(u)-sepharose-selected entamoeba histolytica trophozoite rna in order to clone and identify surface antigens. the library was screened with rabbit polyclonal anti-e. histolytica serum. a 700-base-pair cdna insert was isolated and the nucleotide sequence was determined. the deduced amino acid sequence of the cdna revealed a cysteine-rich protein. dna hybridizations showed that the gene was specific to e. histolytica since the cdna probe reacted w ...19902201027
localization of myosin ic and myosin ii in acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy.polyclonal antisera have been raised against purified acanthamoeba myosin ii and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of acanthamoeba myosin ic. these antisera are specific for their respective antigens as determined by immunoblotting after sds-page of total cell lysates. by using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. myosin ii occurred in the cell cytoplasm ...19902229179
differentiation of naegleria fowleri from acanthamoeba species by using monoclonal antibodies and flow cytometry.monoclonal antibodies to naegleria fowleri and acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured n. fowleri and acanthamoeba spp. four monoclonal antibodies to n. fowleri were specific for n. fowleri and had no reactivity to a. polyphaga. similarly, four monoclonal antibodies to a. polyphaga did not react with n. fowleri. two of the ...19902229384
effect of growth temperature on fatty acid biosynthesis in acanthamoeba castellanii. 19902276479
the effect of currently available contact lens disinfection systems on acanthamoeba castellanii and acanthamoeba polyphaga.contact lens disinfection systems were evaluated for their effectiveness in killing acanthamoeba castellanii and acanthamoeba polyphaga trophozoites and cysts. amoebae were inoculated into commercially available contact lens cleaning and soaking solutions. at intervals varying from 30 minutes to 24 hours, solutions were filtered. the filters were removed and cultured for acanthamoeba organisms. striking differences were observed in the abilities of the different disinfecting solutions to kill th ...19902336265
the covalent structure of acanthamoeba actobindin.actobindin is a protein from acanthamoeba castellanii with bivalent affinity for monomeric actin. because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of f-actin elongation. the complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, staphylococcus v8 protease, endoproteinase asp-n, and cnbr. actobindin contains 2 ...19902376577
rapid diagnosis of acanthamoeba keratitis from corneal scrapings using indirect fluorescent antibody staining.two soft contact lens wearers using a homemade saline solution developed corneal stromal inflammation and epithelial ulceration and were both treated for months with a presumptive diagnosis of herpes simplex keratitis. subsequently, corneal scrapings revealed refractile, cystic structures consistent with the appearance of acanthamoeba. this was rapidly confirmed by indirect fluorescent antibody studies, and acanthamoeba castellani was later identified by growth in culture in both cases. acantham ...19862428344
preliminary characterization of an organism isolated from a case of viluy encephalomyelitis indicates a protozoal, rather than viral, aetiology.a microbial agent was isolated previously from a case of viluy encephalomyelitis and named the 'kpn agent' after the initials of the patient. here a detailed characterization of nucleic acids extracted from the purified kpn agent is presented. the agent contains both dna and rna, and has its own trnas and some other low-mr rnas, including 5s rna. these findings, and the isolation of eukaryotic-type ribosomes, suggest that the kpn agent is not a virus, as believed before, but a more complex micro ...19862428925
rapid visualization of acanthamoeba using fluorescein-conjugated lectins.we investigated the efficacy of fluorescein-conjugated lectins (fcls) for the rapid visualization of acanthamoeba species. cultures of acanthamoeba castellani, acanthamoeba culbertsoni, and acanthamoeba polyphaga were established on nonnutrient agar plates supplemented with escherichia coli. maximal trophozoite populations were established four to five days after initial subculturing; mature cysts were routinely noted three to six days later. at various time points, trophozoites and/or cysts wer ...19882458096
myosin i heavy-chain genes of acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform.we have determined the complete sequence and structure of a second myosin i heavy-chain gene from acanthamoeba castellanii. this gene, which we have named mil, spans approx. 6kb, is split by 17 introns, encodes a 1147-aa polypeptide, and is transcribed in log-phase cells. the positions of six of the introns are conserved relative to a vertebrate muscle myosin gene. similar to the previously characterized mib heavy-chain gene, the deduced mil heavy-chain aa sequence reveals a 125-kda protein comp ...19892511079
cooperative dependence of the actin-activated mg2+-atpase activity of acanthamoeba myosin ii on the extent of filament phosphorylation.the actin-activated mg2+-atpase of myosin ii from acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin ii is active, whereas the phosphorylated and dephosphorylated forms have identical ca2+-atpase activities and mg2+-atpase activities in the absence of f-actin. we have now chemically modified phosphorylated and dephosphorylated myosin ii with n-ethylmaleimide (nem). the modification occ ...19892521858
microinjection into acanthamoeba castellanii of monoclonal antibodies to myosin-ii slows but does not stop cell locomotion.to study the in vivo role of myosin-ii in acanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin-ii heavy chain. all injected cells underwent a transient shock response. it was found that although injection of buffer alone or of an endogenous acanthamoeba protein decreased the motility of injected cells from 7 microns/min to approximately 3 microns/min, injection of monoclonal antibodies specific for myosin-ii decreased motility further to ...19892523248
purification and characterization of a third isoform of myosin i from acanthamoeba castellanii.a third isoform of myosin i has been isolated from acanthamoeba and designated myosin ic. peptide maps and immunoassays indicate that myosin ic is not a modified form of myosin ia, ib, or ii. however, myosin ic has most of the distinctive properties of a myosin i. it is a globular protein of native mr approximately 162,000, apparently composed of a single 130-kda heavy chain and a pair of 14-kda light chains. it is soluble in mgatp at low ionic strength, conditions favoring filament assembly by ...19892530229
membrane carbohydrate characterization of acanthamoeba astronyxis, a. castellanii and naegleria fowleri by fluorescein-conjugated lectins.a comparative study of membrane carbohydrate characteristics of pathogenic and non-pathogenic trophozoites and cysts of free-living acanthamoeba castellanii, naegleria fowleri and a. astronyxis, respectively from sewage sludge in india was carried out by means of fluorescein-conjugated lectin binding using eight lectins. two lectins, viz. concanavalin a and phytohaemagglutinin p, could bind all free-living amoebae at different concentrations. the most notable feature of the study is that peanut ...19892592141
adherence of acanthamoeba castellanii cysts and trophozoites to unworn soft contact lenses.contact lens-related acanthamoeba keratitis has been associated with the use of soft contact lenses and homemade saline solutions. we studied the adherence of acanthamoeba to unworn extended-wear soft contact lenses on a human corneal isolate of acanthamoeba castellanii suspended in normal saline (cysts, 6.3 x 10(5)/ml; trophozoites, 3.6 x 10(5)/ml). unworn hydrogel contact lenses (polymacon, water content 38.6%; 50 lenses, 400 lens segments) were exposed to a. castellanii cysts or trophozoites ...19892596545
antigenic reactivity of a monoclonal antibody against amoeba proteus actin.the reactivity of a monoclonal antibody against actin of amoeba proteus with actins from other sources was examined. the monoclonal antibody cross-reacted with actins from vertebrate muscles, human erythrocytes, and acanthamoeba castellanii, but it did not react with naegleria gruberi actin. the amoeba actin was resolved into 3 bands with isoelectric points of 5.96, 6.03 and 6.10 in electrofocusing gels and they corresponded to 3 peptide spots reacting with the antibody on 2-dimensional immunobl ...19892600879
srrna evolution and phylogenetic relationships of the genus naegleria (protista: rhizopoda).a rapid rna sequencing technique was used to partially sequence the small-subunit ribosomal rna (srrna) of four species of the amoeboid genus naegleria. the extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. however, the pattern of variation among the naegleria species was quite different from that found for those species of tetrapods characterized to date. a phylogenetic analysis of the consensus naegleria ...19892622334
microbiological evaluation of miraflow.the antimicrobial activity of miraflow, an extra-strength cleaner containing 20% isopropyl alcohol, was evaluated using various microorganisms including acanthamoeba. other leading cleaners, disinfecting solutions and heat were evaluated for comparison. miraflow had greater antimicrobial activity than the other cleaning solutions against all five test microorganisms. when evaluated against acanthamoeba castellanii, miraflow was significantly better than disinfecting solutions containing 3% hydro ...19892794328
comparative antimicrobial efficacy of soft and rigid gas permeable contact lens solutions against acanthamoeba.ten soft contact lens cleaners, three rigid gas permeable contact lens (rgp) disinfecting-soaking solutions, and four hydrophilic contact lens disinfecting solutions were evaluated by laboratory challenge procedures for their antimicrobial efficacy against trophozoites and cysts of acanthamoeba polyphaga and acanthamoeba castellanii. miraflow was the only cleaner that killed trophozoites and cysts on all lenses during the cleaning step. a disinfecting solution preserved with chlorhexidine and on ...19892805312
the effects of freezing and antibiotics on the viability of acanthamoeba cysts.the effects of cryotherapy and antibiotics (paromomycin, neomycin, or propamidine isethionate) on the viability of acanthamoeba polyphaga and acanthamoeba castellani cysts were studied in vitro. either cryotherapy or exposure to antibiotic led to a decrease in the number of viable a castellani detected; a polyphaga showed variable response to the antibiotics tested. the combination of cryotherapy and antibiotic therapy was more cysticidal than either modality alone and eliminated detectable viab ...19892923570
glycoprotein phosphorylation in simple eucaryotic organisms. identification of udp-glcnac:glycoprotein n-acetylglucosamine-1-phosphotransferase activity and analysis of substrate specificity.udp-n-acetylglucosamine:glycoprotein n-acetylglucosamine-1-phosphotransferase activity has been identified in both acanthamoeba castellani and dictyostelium discoideum. each of these activities exhibits a different in vitro specificity toward various purified glycoproteins. the n-acetylglucosaminyl-phosphotransferase of a. castellani is very similar to the mammalian enzyme in that it phosphorylates the lysosomal enzymes cathepsin d and uteroferrin much more efficiently than nonlysosomal glycopro ...19862939074
phosphorylation of the oligosaccharide of uteroferrin by udp-glcnac:glycoprotein n-acetylglucosamine-1-phosphotransferases from rat liver, acanthamoeba castellani, and dictyostelium discoideum requires alpha 1,2-linked mannose residues.we have investigated the oligosaccharide requirements of the udp-glcnac:glycoprotein n-acetylglucosamine-1-phosphotransferases from rat liver, acanthamoeba castellani, and dictyostelium discoideum. uteroferrin, an acid hydrolase, was phosphorylated by the three n-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. in all three cases, the phosphorylated species contained 6 or more mann ...19862939075
isolation and partial characterization of a 110-kd dimer actin-binding protein.two triton-insoluble fractions were isolated from acanthamoeba castellanii. the major non-membrane proteins in both fractions were actin (30-40%), myosin ii (4-9%), myosin i (1-5%), and a 55-kd polypeptide (10%). the 55-kd polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. all of these proteins were much more concentrated in the triton-insoluble fractions than in the whole homogenate or soluble supernatant. the 55-kd polypeptide was extracted with ...19862942552
atpase activities and actin-binding properties of subfragments of acanthamoeba myosin ia.previous studies had led to the conclusion that the globular, single-headed myosins ia and ib from acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to f-actin activates the mg2+-atpase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated atpase activity positively cooperative with respect to myosin i concentration. we have now prepared a 100,000-da nh2-terminal pept ...19862946692
extensive purification from acanthamoeba castellanii of a microtubule-dependent translocator with microtubule-activated mg2+-atpase activity.a protein which supported mgatp-dependent movement of latex beads from the minus to the plus end of microtubules and which had microtubule-activated mg2+-atpase was purified from acanthamoeba castellanii. at concentrations as low as 0.6 micrograms ml-1, the translocator supported movement of beads at a rate of 3 to 4 micron s-1. the translocator protein had a ca2+-atpase activity of 1.7 mumol min-1 mg-1 and a mg2+-atpase activity of about 0.03 mumol min-1 mg-1 in the absence of microtubules. the ...19872960674
uv inactivation of pathogenic and indicator microorganisms.survival was measured as a function of the dose of germicidal uv light for the bacteria escherichia coli, salmonella typhi, shigella sonnei, streptococcus faecalis, staphylococcus aureus, and bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus sa11, the cysts of the protozoan acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. the doses of uv light necessary for a 99.9% inactivation of the cult ...19852990336
the ribosomal rna promoter of acanthamoeba castellanii determined by transcription in a cell-free system.the dna sequences required for faithful initiation of ribosomal rna transcription were determined. bal-31 digestion was used to modify the rdna template by introducing deletions from its 3'- and 5'-ends. the resulting mutant dnas were tested for template activity individually or in competition with wild type utilizing an in vitro transcription system from acanthamoeba castellanii. the results identify the sequence extending from -31 to +8 to be absolutely required for transcription. in addition; ...19852995922
in vitro deacylation of lipopolysaccharide of salmonella minnesota by acanthamoeba castellanii enzymes.enzymatic deacylation of the lipopolysaccharide isolated from a salmonella rd mutant by a cell-free preparation from acanthamoeba castellanii has been studied. the degradation was found to be dependent on the presence of a surface-active component (triton x-100) in the reaction mixture. the lipid a part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.e. lauric, myris ...19863007130
genetic evidence that acanthamoeba myosin i is a true myosin.acanthamoeba castellanii contains two enzymes, myosins ia and ib, that exhibit the catalytic properties of a myosin but possess very unusual physical properties, the most striking of which are their single, low molecular weight heavy chain, their globular shape, and their inability to form bipolar filaments. we have now isolated a putative myosin ib heavy chain gene from acanthamoeba, using as a heterologous probe a portion of a sarcomeric myosin heavy chain gene from caenorhabditis elegans. the ...19863014500
complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from acanthamoeba: evidence of a hinge in the rodlike tail.we have completely sequenced a gene encoding the heavy chain of myosin ii, a nonmuscle myosin from the soil ameba acanthamoeba castellanii. the gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. the positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. the deduced myosin ii globular head amino acid sequence shows a high degree of similarity with the globular head sequence ...19873040773
elevated corneal epithelial lines in acanthamoeba keratitis.elevated corneal epithelial lines are another clinical sign in acanthamoeba corneal infection. in this report, one patient wore extended wear soft contact lenses, and another wore daily wear soft contact lenses. both patients used distilled water and salt tablets in their lens care. histopathologic examination of these lines revealed trophozoites and cysts. in one of the patients following penetrating keratoplasty, acanthamoeba castellani and acanthamoeba polyphaga were cultured by impression cy ...19883046583
length variation in eukaryotic rrnas: small subunit rrnas from the protists acanthamoeba castellanii and euglena gracilis.we have sequenced the region of the acanthamoeba castellanii ribosomal rna transcription unit which encodes the mature small subunit ribosomal rna (ssu rrna). it, like the ssu rrna coding regions of euglena gracilis and kinetoplastids, is approx. 30% larger than those reported from other eukaryotes. the extra nucleotides are present in highly variable regions of the rrna genes. direct sequence analysis of the corresponding variable regions in the rrna of a. castellanii and e. gracilis demonstrat ...19863095190
inhibition of multiplication in acanthamoeba castellanii by specific inhibitors of ornithine decarboxylase.proliferation of acanthamoeba castellanii (neff strain) in either a broth medium or a defined medium was arrested by alpha-monofluoromethyldehydroornithine (delta-mfmome), alpha-difluoromethylornithine (dfmo), and (r,r')-delta-methyl-alpha-acetylenic putrescine (map), three specific inhibitors of ornithine decarboxylase. although all three inhibited the ameba enzyme, delta-mfmome was the most effective inhibitor of multiplication. growth inhibition was reversed by the addition of polyamines. the ...19873116220
effect of ribosome-inactivating proteins on ribosomes from tetrahymena pyriformis and acanthamoeba castellanii.the effect of ribosome-inactivating proteins type 1 (single-chain) and type 2 (two-chain, toxins) on polyphenylalanine polymerization by tetrahymena pyriformis and acanthamoeba castellanii ribosomes has been studied. the reaction catalysed by tetrahymena ribosomes was inhibited by two ribosome-inactivating proteins type 1 (dianthin 32 and, less effectively, momordin) whereas the reaction catalysed by amoeba ribosomes was inhibited, in a decreasing order of activity, by three ribosome-inactivatin ...19873120707
events during eucaryotic rrna transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.chemical footprinting and topological analysis were carried out on the acanthamoeba castellanii rrna transcription initiation factor (tif) and rna polymerase i complexes with dna during transcription initiation and elongation. the results show that the binding of tif and polymerase to the promoter does not alter the supercoiling of the dna template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted dna regions. thus, in contrast to bacte ...19883133551
filament formation and actin-activated atpase activity are abolished by proteolytic removal of a small peptide from the tip of the tail of the heavy chain of acanthamoeba myosin ii.actin-activated mg2+-atpase activity of myosin ii from acanthamoeba castellanii is regulated by phosphorylation of three serine residues located at the carboxyl-terminal end of each of the two 185,000-da heavy chains; the phosphorylated molecule has full ca2+-atpase activity but no actin-activated mg2+-atpase activity. under controlled conditions, chymotrypsin removes a small peptide containing all three phosphorylation sites from the ends of the myosin ii heavy chains producing a molecule with ...19853155741
purification from dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin i from acanthamoeba castellanii.a low-molecular-weight myosin has been purified 1500-fold from extracts of dictyostelium discoideum, based on the increase in k+,edta-atpase specific activity. the purified enzyme resembles the single-headed, low-molecular-weight myosins ia and ib from acanthamoeba castellanii, and differs from the conventional two-headed, high-molecular-weight myosin previously isolated from dictyostelium, in several ways. it has higher k+,edta-atpase activity than ca2+-atpase activity; it has a native molecula ...19853157680
detection of glycoproteins in the acanthamoeba plasma membrane.in the present study we have shown that glycoproteins are present in the plasma membrane of acanthamoeba castellanii by utilizing different radioactive labeling techniques. plasma membrane proteins in the amoeba were iodinated by 125i-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. the periodate/nab3h4 and galactose oxidase/nab3h4 labeling techniques were used for la ...19883169144
purification of plasma membrane from acanthamoeba castellanii.a simple method for isolation of plasma membrane from acanthamoeba using self-generating gradients of percoll is described. to obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. the purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. as judged from enrichment of radiolabel, a 37-fold pu ...19883184000
crystallization of actophorin, an actin filament-severing protein from acanthamoeba.actophorin is an actin monomer-binding and actin filament-severing protein from acanthamoeba castellanii. it crystallizes out of polyethylene glycol in a form suitable for high resolution x-ray analysis. the crystals are orthorhombic and have the symmetry of the space group p2(1)2(1)2(1) with lattice constants a = 39.8 +/- 0.5, b = 47.3 +/- 0.6, and c = 69.9 +/- 1.6 a. they diffract to a resolution of at least 2.8 a, and the asymmetric unit contains one actophorin monomer of mr 15,000.19883192529
resolution of acanthamoeba castellanii chromosomes by pulsed field gel electrophoresis and construction of the initial linkage map.pulsed field gel electrophoresis has been used to resolve chromosome-sized dna molecules in fungi and parasites but has not yet been used successfully to examine the chromosomes of other lower eukaryotes used extensively for biochemical research such as acanthamoeba, physarum, and dictyostelium. here we show an electrophoretic karyotype of the protozoan acanthamoeba castellanii using orthogonal field alternating gel electrophoresis (ofage). there are about 20 small chromosomes ranging in size fr ...19883219918
survival of coliforms and bacterial pathogens within protozoa during chlorination.the susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of acanthamoeba castellanii and tetrahymena pyriformis. cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated ...19883223766
concanavalin a-induced redistribution of surface receptors in acanthamoeba castellanii at different growth phases.concanavalin a (cona)-induced redistribution of surface receptors has been studied in acanthamoeba castellanii at different growth phases utilizing double fluorescent techniques and transmission electron microscopy. when the amoebae were incubated with 2 micrograms and 10 micrograms tetramethylrhodamine isothiocyanate (tritc)-cona/ml for 4 min and 15 min at 28 degrees c the staining pattern was characterized by various numbers of scattered aggregates of fluorescent cona. double labeling of the a ...19883229417
relationship between the timing of dna replication and the developmental competence in acanthamoeba castellanii.in acanthamoeba, two different cell types are known. trophozoites are generated in the mitotic division cycle, whereas cells committed at late g2 phase of the cell cycle develop into cysts in response to starvation. in this paper we study the role of timing of dna replication in regulating development. the investigation was performed with cultures growing in a non-defined medium (nd cells) that show a high encystation competence and with cultures that have been growing in a chemically defined me ...19883256538
production of monoclonal antibodies to naegleria fowleri, agent of primary amebic meningoencephalitis.monoclonal antibodies (mabs) to naegleria fowleri, the etiologic agent of primary amebic meningoencephalitis (pam), have been produced and used as probes to identify n. fowleri amebae in brain sections of patients who died of that disease. these mabs were characterized for their specificity by the indirect immunofluorescence assay (iif), dot immunobinding assay (diba), and enzyme-linked immunotransfer blot technique (eitb). the mabs reacted intensely with all strains of n. fowleri tested origina ...19873308948
acanthamoeba keratitis and infectious crystalline keratopathy.two cases of acanthamoeba keratitis and infectious crystalline keratopathy, occurring simultaneously, are presented. three and 12 months after initiating topical corticosteroid therapy in cases 1 and 2, respectively, alpha-hemolytic streptococcus viridans was cultured from each cornea. topical corticosteroid therapy was initiated for the treatment of an annular stromal opacity, presumably secondary to herpes simplex keratitis. acanthamoeba was identified in culture following penetrating keratopl ...19873314824
distribution and cellular localization of actin depolymerizing factor.actin depolymerizing factor (adf) is a low molecular mass (19 kd) protein that forms a tightly bound dimeric complex with actin. we have raised a rabbit antiserum to chick brain adf and used it to analyze the distribution and cellular localization of adf. we find that adf is a major constituent of all chick embryonic and most adult tissues examined, accounting for 0.1-0.4% of the total protein. some tissues have as much as 0.6 mol adf per mole actin. adult heart and skeletal muscle are unusual i ...19873320057
a temperature-compensated ultradian clock explains temperature-dependent quantal cell cycle times.the effects of sublethal heat pulses on cell division have provided insights into possible molecular mechanisms. thus zeuthen's findings of 'set-backs' up to a transition point provides the basis for the idea that the continuous accumulation of a compound needed for cell division spans a major portion of the cell cycle. the accumulating substance is a 'division protein' which forms part of a structure which is unstable until completely assembled at the transition point. experiments showing phase ...19873332482
effects of single-base substitutions within the acanthamoeba castellanii rrna promoter on transcription and on binding of transcription initiation factor and rna polymerase i.single-point mutations were introduced into the promoter region of the acanthamoeba castellanii rrna gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. the promoter mutants were tested for transcription initiation factor (tif) binding by a template commitment assay plus dnase i footprinting and for transcription by an in vitro runoff assay. point mutations within the previously identified tif interaction ...19883352603
inhibition of an early stage of actin polymerization by actobindin.actobindin, a 25,000-dalton dimeric protein purified from acanthamoeba castellanii was previously shown to form a 1:1 molar complex with both acanthamoeba and rabbit muscle g-actin with kd values of about 5 and 7 microm, respectively, and not to interact with f-actin (lambooy, p. k., and korn, e. d. (1986) j. biol. chem. 261, 17150-17155). we now find that actobindin is a much more potent inhibitor of the early phases of polymerization of both acanthamoeba and muscle g-actin than can be accounte ...19883417638
purine salvage by acanthamoeba castellanii. 19873429118
the heavy chain of acanthamoeba myosin ib is a fusion of myosin-like and non-myosin-like sequences.acanthamoeba castellanii myosins ia and ib demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. we now present the complete amino acid sequence of the 128-kda myosin ib heavy chain, which we deduced from the nucleotide sequence of the gene and which reveals that the ...19873477803
effect of essential amino acids on the phosphorylation of a 40s ribosomal protein and protein synthesis in acanthamoeba castellanii.reversible and multiple phosphorylation of a 40s ribosomal protein is observed in a variety of eukaryotic cells. in the primitive eukaryote acanthamoeba, one or three phosphorylated s3 derivatives are observed during growth phase in nondefined nutrient medium (nd cells) or in chemically defined nutrient medium (d cells), respectively. in both cases, stationary phase cells exhibit nonphosphorylated s3; however, transfer of these cells into the respective fresh nutrient media results in a transien ...19873558495
isolation and identification of 1(3),2-diacylglyceryl-(3)-o-4'-(n,n,n-trimethyl)homoserine from the soil amoeba, acanthamoeba castellanii.a polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan acanthamoeba castellanii. on the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (dgts). fast atom bombardment (fab) mass spectra of dgts are reported for the first time and are compared to the fab mass spectra of phosphatidylcholines and the electron ionization (ei) and field desorption (fd) mass spectra of dgts. ga ...19863559384
effect of phalloidin and viroisin on acanthamoeba castellanii after permeabilization of the cell.we have developed a new technique for the permeabilization of the membrane of acanthamoeba castellanii. this technique involves the use of digitonin which alters neither the morphology nor the motility of the cell, but favours the penetration of phalloidin and viroisin. treatment of permeabilized cells with phalloidin or viroisin induces, in the cortex of the cell, an intensive proliferation of filaments which have been identified as actin. this cortical filamentous layer detaches from the membr ...19873580176
temperature-compensated ultradian variation in cellular protein content of acanthamoeba castellanii revisited.synchronous cultures of the soil amoeba acanthamoeba castellanii, established in the laboratory by selection procedures, show oscillations of total cell protein, as well as respiration. previously, time series analysis by aspect, a university of warwick computer unit program, showed an average period of 76 min. a reanalysis by least-squares rhythmometry revealed that, in general, the analyses accompanying the original paper and those in this replication agree very well. the periods extracted by ...19873601955
polyamine metabolism in acanthamoeba: polyamine content and synthesis of ornithine, putrescine, and diaminopropane.five polyamines which could be separated by high performance liquid chromatography were found in acanthamoeba castellanii (strain neff). these included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. only diaminopropane and norspermidine had been found previously. spermine was present in cultures grown in broth, but not in defined medium. radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ...19873656216
identification of a new type of mammalian myosin heavy chain by molecular cloning. overlap of its mrna with preprotachykinin b mrna.in this paper, we report the polypeptide sequence, the mrna sequence, and gene organization of a new category of mammalian myosin heavy chain, termed myosin i heavy chain-like protein (mihc), on the basis of sequence analyses of cdna clones isolated from a bovine intestinal cdna library and a genomic dna clone containing the mihc gene. the mihc mrna shares its 3' sequence with the 5' sequence of the preprotachykinin b mrna encoding the precursor for the neuromedin k neuropeptide. an overlap of t ...19873667594
propulsion of organelles isolated from acanthamoeba along actin filaments by myosin-i.eukaryotic cells are dependent on their ability to translocate membraneous elements about the cytoplasm. in many cells long translocations of organelles are associated with microtubules. in other cases, such as the rapid cytoplasmic streaming in some algae, organelles appear to be propelled along actin filaments. it has been assumed, but not proven, that myosin produces these movements. we have tested vesicles from another eukaryotic cell for their ability to move on the exposed actin bundles of ...19863748157
crystallization of acanthamoeba profilin-i.profilin-i, a protein that inhibits actin polymerization in acanthamoeba castellanii, has been crystallized in a form suitable for high resolution x-ray analysis. the crystals have the symmetry of the space group c2 with lattice constants a = 110.4 +/- 0.2, b = 31.7 +/- 0.1, c = 33.5 +/- 0.1 a, beta = 112.2 degrees. they diffract to at least 2.0-a resolution. the asymmetric unit contains one 12,800-dalton monomer of profilin-i.19863759969
purification and characterization of actobindin, a new actin monomer-binding protein from acanthamoeba castellanii.actobindin is a new actin-binding protein isolated from acanthamoeba castellanii. it is composed of two possibly identical polypeptide chains of approximately 13,000 daltons, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and with isoelectric points of 5.9. in the native state, actobindin appears to be a dimer of about 25,000 daltons by sedimentation equilibrium analysis. it contains no tryptophan and probably no tyrosine. actobindin reduces the concentration of f-ac ...19863782158
amoebae isolated from the atmosphere of mexico city and environs.a protozoological analysis was performed from june to august, 1982 to isolate small free-living amoebae from the atmosphere by using an air vacuum sampler and several filters. monoxenic medium (nonnutritive agar plus escherichia coli) and axenic medium (de jonckheere, 1977) were used to culture the isolates. the species isolated included naegleria sp. alexeieff emend. calkins, acanthamoeba polyphaga puschkarew, vahlkampfia jugosa page, acanthamoeba astronyxis ray and hayes, acanthamoeba castella ...19873803333
purification and characterization of an atp-sensitive actin gelation protein from acanthamoeba castellanii.a protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from acanthamoeba castellanii. this protein, gf-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 da. as determined by the method of falling ball vicometry, gf-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. actin gels did not form in the pr ...19873818648
a versatile perfusion technique for metabolic studies by nmr.a perfusion technique applicable to metabolic studies by nmr is described in detail. the utility and versatility of the approach are demonstrated by following gluconeogenesis from [2-13c]pyruvate in the perfused, isolated mouse liver, and lipogenesis from [2-13c]acetate in perfused acanthamoeba castellanii cells embedded in agarose filaments.19853831678
acanthamoebic keratitis in a healthy australian man.we report a case of acanthamoebic keratitis that occurred after minor ocular trauma in a healthy 31-year-old man. multiple microbiological investigations failed to reveal the causative organism, which was identified as acanthamoeba castellanii only after a corneal graft operation had been performed. a review of previously described cases reveals that this rare ocular infection tends to cause recalcitrant corneal ulcers after minor eye injuries in otherwise healthy individuals.19853831753
ribosomal rna transcription: proteins and dna sequences involved in preinitiation complex formation.an in vitro transcription system consisting of partially purified transcription initiation factor(s) and purified rna polymerase i from acanthamoeba castellanii was used to study the mechanism of faithful initiation of ribosomal rna transcription. formation of a preinitiation complex between one or several auxiliary transcription proteins and the dna template in the absence of rna polymerase i was demonstrated. a series of 3'- and 5'-deletion mutants of the template was used in prebinding compet ...19853856847
a prominent microtubule cytoskeleton in acanthamoeba.a method for preparing by detergent extraction the cytoskeletons of substrate-attached, motile acanthamoeba castellanii is described. a monoclonal antibody to yeast alpha tubulin has been used to demonstrate the presence of abundant microtubules in the cytoskeleton of this amoeba by fluorescence and whole-mount electron microscopy. individual microtubules, often more than 10 micron long, interweave to form a well-developed 3-d network pervading the cytoplasm and embracing the nucleus. in some ca ...19853888418
oxidative metabolism associated with phagocytosis in acanthamoeba castellanii.when amebae were incubated with latex beads, cyanide-insensitive oxygen consumption increased nearly two-fold. this cyanide-insensitive respiration was inhibited by salicylhydroxamate. furthermore, cell fractionation studies revealed a localization for a portion of the nad(p)h oxidase activity in phagolysosomes. the presence of low concentrations of divalent metal during fractionation resulted in an increased yield of oxidative activity in the phagolysosome fraction. in addition, the phagolysoso ...19853925134
polyphenol oxidase produced during encystation of acanthamoeba castellanii.acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. acanthamoeba polyphenol oxidase has a ph optimum of 6.0 and a km value of 0.21 mm with dihydroxyphenylalanine. the enzyme does not oxidize tyrosine, and it is inhibited by c ...19853930706
amebic meningoencephalitis caused by acanthamoeba castellani in a dog.a natural infection of acanthamoeba castellani, a free-living ameba, was determined to be the cause of acute, hemorrhagic, necrotizing amebic meningoencephalitis in a dog. this case is unique because previous reports of infection by the acanthamoeba spp in dogs have not indicated its presence in the brain. naturally developing meningoencephalitis by acanthamoeba spp in the dog may have a pathogenesis similar to that of human beings. the ameba in this case also was observed in the lungs and kidne ...19853932273
purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from acanthamoeba castellanii.actophorin is a new actin-binding protein from acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. the isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. the phosphate content is less than 0.2 mol/mol. there is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. unique two-dimensional maps of tryptic and chymotryptic peptides and comp ...19863941084
amino acid sequence of the active site of acanthamoeba myosin ii.we have used the substrate [5,6-3h]utp for direct photoaffinity labeling of the active site of the heavy chain of myosin ii from acanthamoeba castellanii. the only labeled peptide in a total tryptic digest had the sequence of thr-glu-asn-thr-me2lys-lys (where me2lys represents dimethyllysine) with the substrate covalently bound to the glu residue. this sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (me2lys----lys), a post-translational ...19863944113
isolation of a non-muscle myosin heavy chain gene from acanthamoeba.we have isolated a non-muscle myosin heavy chain gene from acanthamoeba castellanii using as a heterologous probe a sarcomeric myosin heavy chain gene from caenorhabditis elegans. the amoeba genomic clone has been tentatively identified as containing a myosin ii heavy chain gene based on hybridization to a 5300-nucleotide rna species, hybrid selection of a mrna encoding a 185-kda polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin ii, and an exact match between ...19863944121
pathogenic and free-living protozoa cultured from the nasopharyngeal and oral regions of dental patients: ii.protozoa of the nose, mouth, and pharynx of 30 randomly chosen male caries patients at an odontological clinic of the national autonomous university of mexico, in mexico city, were surveyed by culture from swabs. culture tubes from swabs were observed every other day for 5 weeks. pathogens found included entamoeba histolytica, naegleria fowleri, acanthamoeba castellanii, a. culbertsoni, a. polyphaga, and giardia lamblia. such isolations of pathogens suggest that patients may be healthy carriers ...19863956463
susceptibility of acanthamoeba to soft contact lens disinfection systems.members of the genus acanthamoeba are increasingly recognized as agents of indolent, chronic, infectious keratitis. recently, acanthamoeba corneal infection has been reported in some persons who wear soft contact lenses. in this study, three "heat" and three "cold" soft contact lens disinfection systems were tested according to the manufacturers' instructions against acanthamoeba castellanii and acanthamoeba polyphaga in separate trials, and with appropriate controls. suspensions of acanthamoeba ...19863957582
[incidence of acanthamoeba castellanii meningoencephalitis in hungary]. 19854047649
lack of nh2-terminal processing of actin from acanthamoeba castellanii.acanthamoeba actin is the only actin sequenced to date that has neither an nh2-terminal ac-asp nor ac-glu residue. the protein begins with an ac-gly-asp and is coded for by a gene that specifies a polypeptide beginning met-gly-asp. thus, the acanthamoeba actin gene would appear to specify a class ii actin with the usual nh2-terminal cys replaced with a gly. previous studies (rubenstein, p. a., and martin, d. j. (1983) j. biol. chem. 258, 11354-11360) revealed that for class ii actins the met is ...19854055803
differentiation in acanthamoeba castellanii is induced by specific monoclonal antibodies.monoclonal antibodies that bind a large molecular weight plasma membrane protein of acanthamoeba castellanii cause the cells to differentiate. a different monoclonal antibody that binds specifically to the major plasma membrane protein has no effect upon cell division or differentiation. the induction of differentiation by the monoclonal antibodies requires a bivalent attachment, more than a single binding cycle of the antibody to the plasma membrane protein, does not require cell-cell contact, ...19854086510
isolation of nuclei and characterization of nuclear dna of acanthamoeba castellanii. 19744139978
[properties of the mitochondrial electron transport system of acanthamoeba castellanii neff. i. effect of inhibitors on various acceptor systems tested by means of a modified thunberg technic]. 19734145409
[properties of the mitochondrial electron transport system of acanthamoeba castellanii neff. ii. purification and properties of the nadh-acceptor-oxidoreductase of the electron transport system]. 19734145410
carbohydrate metabolism in acanthamoeba castellanii. ii. carbon dioxide fixation reactions. 19744151330
acanthamoeba myosin. i. isolation from acanthamoeba castellanii of an enzyme similar to muscle myosin. 19734268863
effect of 5-bromodeoxyuridine on growth, encystment, and excystment of acanthamoeba castellanii. 19744274263
lipids of acanthamoeba castellanii. composition and effects of phagocytosis on incorporation of radioactive precursors.the lipids of acanthamoeba castellanii (neff) consist of 52% neutral lipids and 48% polar lipids. triglycerides account for 75% and free sterols for 17% of the neutral lipids. the major phospholipids are phosphatidylcholine (45%), phosphatidylethanolamine (33%), phosphatidylserine (10%), a phosphoinositide (6%), and diphosphatidylglycerol (4%). the phosphoinositide is unique in that it contains fatty acids, aldehyde, inositol, and phosphate in the ratio of 1.4:0.5:1.1, but it contains no glycero ...19694309952
phagocytosis of latex beads by acahamoeba castellanii (neff). 3. isolation of the phagocytic vesicles and their membranes.a method is described for the rapid and efficient isolation of phagocytic vesicles from large scale cultures of acanthamoeba castellanii (neff) that have been incubated with polystyrene latex beads. cells were allowed to phagocytose latex beads for 30 min and then were homogenized, and the phagocytic vesicles were isolated by one centrifugation through several layers of sucrose. identity and purity of the phagocytic vesicles were determined by electron microscopy, chemical analyses, and assays o ...19694309954
cell size, macromolecule composition, nuclear number, oxygen consumption and cyst formation during two growth phases in unagitated cultures of acanthamoeba castellanii. 19694312005
ultrastructural characterization of f-actin isolated from acanthamoeba castellanii and identification of cytoplasmic filaments as f-actin by reaction with rabbit heavy meromyosin. 19704318206
plasma and phagosome membranes of acanthamoeba castellanii.plasma membranes were isolated from the ameba acanthamoeba castellanii by low-speed velocity centrifugation followed by equilibrium centrifugation in a sucrose gradient. the isolated membranes had a high ratio of sterol to phospholipid (0.98 moles/mole) and of phospholipid to protein (0.43 mg/mg). the plasma membranes had very low concentrations of dna, rna, lipid inositol, and glycerides. glycolipids and glycoproteins were enriched in the plasma membranes relative to their concentrations in the ...19714329520
exocytosis of latex beads during the encystment of acanthamoeba.cells of acanthamoeba castellanii (neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. the encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase a ...19724331294
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