Guinea Pig Erythrocytes Research Articles

Influence of <i>Yersinia pestis</i> with different plasmid composition on the erythrocyte membrane in the blood of guinea pigs

Introduction. Based on data on the role of blood erythrocytes in the development and implementation of the vaccine and infectious processes in plague, it was of interest to evaluate changes in erythrocyte surface architecture from the position of searching for informative criteria for the preclinical evaluation of anti-plague vaccines. Aim — using atomic force microscopy to characterize the state of the blood erythrocyte membrane of guinea pigs in response to subcutaneous administration of the vaccine strain Yersinia pestis EV NIIEG and its isogenic derivatives. Materials and methods. For immunization of animals strain Y. pestis EV NIIEG (pYT+, pYV+, pYP+) and its isogenic derivatives Y. pestis KM216 (pYT–, pYV–, pYP+), Y. pestis KM217 (pYT–, pYV+, pYP–), Y. pestis KM218 (pYT–, pYV–, pYP–) were used. Analysis of the erythrocyte membrane was carried out using a Solver P47-PRO scanning probe microscope (NT-MDT, Russia). Results. The most pronounced changes in the surface architectonics of the membrane of guinea pig erythrocytes were established during the first three days of the formation of the immune response to the Y. pestis EV strain NIIEG and its isogenic variant Y. pestis KM217, in the genome of which the pYV plasmid is preserved, administered at a dose of 5 × 108 CFU. A significant increase (p 0.05) in the proportion of transformed cell forms (43.67 ± 3.63% and 37.83 ± 7.03% versus 4.08 ± 0.86% in the control group), root mean square roughness (319 ± 8 nm and 312 ± 7 nm versus 70 ± 6 nm in the control group), Young's modulus (125.73 ± 4.48 kPa and 113.8 ± 5.41 kPa versus 53.03 ± 1.47 kPa in the control group). By the 21st day, the value of these indicators decreased by an average of 2.7, 2.0 and 1.5 times, respectively, indicating restoration of the erythrocyte membrane. Conclusion. The dependence of the changes in the erythrocyte membrane and the rate of their restoration on the plasmid composition of Y. pestis strains has been established. The data obtained contribute to the understanding of the processes of interaction of Y. pestis with the erythrocyte membrane and can be used as additional characteristics in the development of new criteria for preclinical evaluation of plague candidate vaccines.

The Fis Nucleoid Protein Negatively Regulates the Phase Variation fimS Switch of the Type 1 Pilus Operon in Enteropathogenic Escherichia coli.

In Escherichia coli the expression of type 1 pili (T1P) is determined by the site-specific inversion of the fimS ON–OFF switch located immediately upstream of major fimbrial subunit gene fimA. Here we investigated the role of virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the fimS switch in the human enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the fimS switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆fis mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the fimA, fimB, and fimE promoters using lacZ transcriptional fusions indicated that only PfimA activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the fimS switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of E. coli.

Investigation of Kluyveromyces marxianus as a novel host for large\u2010scale production of porcine parvovirus virus\u2010like particles

BackgroundPorcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines.ResultsAfter optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice.ConclusionsThis is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.

Hemagglutinating properties of bovine parainfluenza-3, coronavirus and rotavirus infections’ pathogens in cattle

The purpose of the work was to determine the hemagglutinating activity of different strains of bovine parainfluenza-3, coronavirus and rotavirus infections’ pathogens in cattle. Accumulation of hemagglutinins of viruses was carried out by infecting cell cultures with viruses, determining their infectious activity by cytopathic action, followed by the establishment of hemagglutinating activity with erythrocytes of different species of animals. It has been established that within one type of virus there may be strains that have different hemagglutinating activity. It has been proved that within one type of virus there may be strains that have different hemagglutinating activity. It has been established that for the detection of coronavirus hemagglutinins mouse erythrocytes are the most suitable, bovine parainfluenza-3 virus hemagglutinins — guinea pig erythrocytes, rotavirus hemagglutinins — rooster erythrocytes. The relationship between the infectious activity of parainfluenza-3 virus, corona-, rotaviruses and their hemagglutinating properties has been established — the highest hemagglutinin titer was observed under the conditions of infectious activity of bovine parainfluenza-3 virus, corona-, rotaviruses in 7.0 lg TCD50/cm3 and higher. Long-term storage of bovine parainfluenza-3, coronavirus and rotavirus infections’ pathogens at a temperature of minus 18 °С and lower did not have a negative effects on their hemagglutinating properties, in contrast to the storage of these viruses at a temperature of minus 4 °С or repeated defrosting

Identification and genetic characterization of bovine parainfluenzavirus type 3 genotype c strain isolated from cattle in western China

Bovine parainfluenza virus type 3 (BPIV3) is a leading respiratory pathogen of cattle and is often implicated as a causative pathogen of bovine respiratory disease complex (BRDC). Three genotypes (BPIV3a-c) have been tentatively classified based on genetic and phylogenetic analyses. A BPIV3c isolate, designated NX49, was isolated using the Madin-Darby bovine kidney cell culture from cattle in western China, where feedlot cattle with severe respiratory disease symptoms were observed. Hemagglutination assay demonstrated that NX49 had low agglutination activity against guinea pig erythrocytes, whereas SD0835, the first BPIV3c strain reported in China, showed high agglutination activity. Phylogenic analysis indicated that NX49 shared 99.3% whole genome identity with SD0835 and 97.5%-98% similarity with BPIV3c strains isolated from Japan, South Korea, and the United States. NX49 also showed 82.5% nucleotide similarity with NM09, a BPIV3a strain isolated from China. These data suggest that the BPIV3c strains circulating in China are conserved. Moreover, BPIV3a and BPIV3c might concomitantly circulate in feedlot cattle for long durations in China. Further characterizing of such viruses and continued surveillance may help to prevent BRDC caused by BPIV3 and aid in vaccine development.

Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract.

Uropathogenic Escherichia coli (UPEC) adhere to cells in the human urinary tract via type 1 pili that undergo phase variation where a 314-bp fimS DNA element flips between Phase-ON and Phase-OFF orientations through two site-specific recombinases, FimB and FimE. Three fim-lux operon transcriptional fusions were created and moved into the clinical UPEC isolate NU149 to determine their temporal regulation in UPEC growing in the urinary tract. Within murine urinary tracts, the UPEC strains demonstrated elevated transcription of fimA and fimB early in the infection, but lower transcription by the fifth day in murine kidneys. In contrast, fimE transcription was much lower than either fimA or fimB early, increased markedly at 24 h after inoculation, and then dropped five days after inoculation. Positioning of fimS was primarily in the Phase-ON position over the time span in UPEC infected bladders, whereas in UPEC infected murine kidneys the Phase-OFF orientation was favored by the fifth day after inoculation. Hemagglutination titers with guinea pig erythrocytes remained constant in UPEC growing in infected murine bladders but fell substantially in UPEC infected kidneys over time. Our results show temporal in vivo regulation of fim gene expression in different environmental niches when UPEC infects the murine urinary tract.

Mycoplasma pneumoniae, A Tale of 50 Years.

The bacteriological study of the nasopharyngeal, throat, per nasal tracheal aspirate, and percutaneous lung aspirate was non-contributory, and virological examinations failed to yield a viral agent. Meanwhile the child received treatment with penicillin and streptomycin for 8 days. She became afebrile on 7th day of hospitalization, and her respiratory findings, adenopathy and splenomegaly disappeared while the liver receded to 2 cm below costal margin. The throat swab specimen after being stored at –20 oC to –70 oC for 27 days was examined for M. pneumoniae by inoculating on agar plates and diphasic medium, and its growth was confirmed on the 7th day of first agar subculture. On day 14 of incubation in diphasic medium, a drop in pH (glucose fermentation) and spherular colonies were observed floating in the media. The colonies demonstrated guinea pig erythrocyte hemolysis (within 48 hours) and sheep cell haemadsorption, properties peculiar to M. pneumoniae. Isolation of M. pneumoniae from throat and tracheal aspirates of this child with pneumonia – whose extensive bacterial and viral investigations were non-contributory – indicated etiological role of M. pneumoniae. The child was treated with demethyl-chlortetracycline. A repeat X-ray 3 month later showed persisting middle lobe infiltrates, although child was asymptomatic.

Effects of Benzoic Acid and its Analogues on Osmotic Fragility in Guinea Pig Erythrocytes In Vitro

In our previous report, benzoic acid and some of its chemical analogues were shown to increase osmotic fragility (OF) in isolated rat red blood cells (RBCs). However, there have been no reports on the effects of benzoic acid and its derivatives on OF in the RBCs of other animals. To clarify these inter-species differences, the effects of those chemicals on OF were examined in guinea pig RBCs. The isolated RBCs were exposed to the chemicals at a concentration of 0.1-100 mM for 1 h. OF was measured by determining the 50% hemolysis of RBCs using a 0.1-0.8% NaCl solution. Benzoic acid and some of its derivatives decreased OF in a dose dependent manner. Replacement of the carboxylic group with another group or the introduction of another element on the benzene ring also affected OF. These effects were dependent on both the type of molecule and the position at which they were introduced into the benzene nucleus. The introduction of chlorine, bromine or iodine tended to increase OF, even at low concentrations. With regard to the action of the tested chemicals, the hydrophobic benzene ring is thought to enter the phospholipid layer while the hydrophilic carboxylic group remains at the membrane surface, perturbing the RBC membrane. Inter-species differences between rat and guinea pig were confirmed in the response of the RBC membrane to benzoic acid and its derivatives. These differences are speculated to be due to differences in the lipid composition of the RBC membrane.

Monocarboxylic Acids and Dicarboxylic Acids Induce Different Responses in terms of Osmotic Fragility in Rat and Guinea Pig Erythrocytes In vitro

We compared the effects of monocarboxylic and dicarboxylic acids on osmotic fragility (OF) in rat and guinea pig red blood cells (RBCs) in vitro. Monocarboxylic acids possessing 4 to 8 straight-chain hydrocarbons increased OF in rat RBCs in a concentration-dependent manner. The increases in OF were also dependent on the number of carbons in the hydrocarbon chain bonded to the carboxylic group. Benzoic and cyclohexane-monocarboxylic acids also increased OF in rat RBCs in a dose-dependent manner. Although most of the dicarboxylic acids possessing a straight hydrocarbon chain decreased OF in rat RBCs, malonic acid showed the greatest potential to decrease OF among the substances tested. None of the monocarboxylic acids increased OF in guinea pig RBCs. Among the dicarboxylic acids possessing a benzene ring, isophthalic and terephthalic, but not phthalic acid, decreased OF in rat RBCs dose-dependently. Three cyclohexane-dicarboxylic acids decreased OF in guinea pig RBCs, while they had no effect on OF in rat RBCs. With regard to the action of those carboxylic acids on the cell membrane, hydrophobic hydrocarbons of a certain structure are thought to enter the phospholipid layer with the hydrophilic carboxylic group remaining at the membrane surface where it affects the nature of the cell membrane, thus changing osmotic resistance in the RBCs. Inter-species differences in the RBC membrane were confirmed in the OF response to those carboxylic acids. The differences in OF response to the monocarboxylic and dicarboxylic acids observed in rat and guinea pig RBCs are speculated to be due to differences in the nature of the RBC membrane formed by the various phospholipids.

Comparison of Inhibitory Activities of meta and para Substituted N-aryl 3-Hydroxypyridin-4-one Mannosides Towards Type 1 Fimbriated E. coli

In uropathogenic Escherichia coli, mannose- specific adhesion is mediated by the FimH adhesin located at the tip of type 1 fimbriae. Novel mannosylated N-aryl-substituted 3- hydroxypyridin-4-ones with meta substituents on the aryl part of the molecule were prepared, and their inhibitory properties towards the adhesion of E. coli to guinea pig erythrocytes explored using the hemagglutination assay. These results were compared with inhibitory potencies of analogous para derivatives. The assays revealed greater preference of FimH towards para substituted compounds in general, with p-nitro and p-methoxy substituted substrates being much more effective then the hydrophobic p-methyl compound. When substituents are in meta position the positive affect on the binding of compounds in the FimH binding site was observed with all compounds tested but the structure with an alkyl group was shown to be the most effective one. This study provides guidelines for the rational design of novel, more effective series of FimH antagonists.

Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast.

BackgroundEleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications.ResultsRecombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25–35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not.ConclusionsThe yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed.

Isolation of novel bovine parainfluenza virus type 5 (bPIV5) and its incidence in Korean cattle

Abstract : Four viruses showing cytopathic effects in MDBK cells were isolated from brains of cattle showing downercattle syndrome in 2012. The isolates were confirmed to belong to the genus Rubulavirus of the subfamilyParamyxovirinae. Isolate QIA-B1201 had the ability to hemagglutinate red blood cells from several species of animalsand was capable of adsorbing guinea pig erythrocytes on the surface of infected Vero cells. Nucleotide sequence analysisshowed that two isolates (QIA-B1201 and QIA-B1204) had high similarity with other human and animal PIV5 isolatesranging from 98.1 to 99.8%. The highest sequence similarity of the two isolates corresponded to strain KNU-11 (99.8%at the nucleotide and amino acid level) isolated from suckling piglets in Korea in 2012. To evaluate the virulenceof strain QIA-B1201, we inoculated bPIV5 into 5 week-old mice via both the intraperitoneal and intracranial route.Body weight was not significantly altered in mice inoculated with QIA-B1201. In this study, we isolated andcharacterized novel bPIV5s from brain samples showing downer cattle syndrome, but were not able to elucidate thepathogenicity of the bPIV5s in mice.Keywords : bovine, neurologic disease, parainfluenza virus 5, virus isolation

Новий манозоспецифічний лектин із кореневищ лілійника рудуватого (Hemerocallis fulva L.): очищення та властивості

A new lectin was purified from the daylily (Hemerocallis fulva L.) with the yield of approximately 10 mg per kg of fresh plant rhizome. The purification procedure was based on application of the affinity chromathography on the column with yeast mannan and the ion-exchange chromatography on the column with DEAE-Toyopearl. The lectin possessed low affinity for alpha-methyl-D-mannopyranoside, D-fructose, D-turanose and 2-acetamido-D-galactopyranose and hight affinity for the yeast mannan. The lectin bound with greatly less affinity for the mannose-containig glycoproteins, such as ovoalbumin, ovomucoid and horseradish peroxidase. According to the results of electrophoresis in 20% DSNa-PAGE, the lectin consists of subunits of 12 kDa molecular weight. According to the results of gel-chromatography on the Toyopearl HW-55, the lectin's molecular weight is 48 kDa. It agglutinated rabbit erythrocytes very well, while rat and guinea-pig erythrocytes were agglutinated worse, and human erythrocytes were not agglutinated at all. Lectin's dialysis against 1% EDTA or heating to 60 degrees C for 60 min did not stop its hemagglutinating activity.

Hemagglutinating activity is directly correlated with colonization ability of shigellae in suckling mouse model

The aim of the present study was to explore a new approach based on the hemagglutination (HA) assay to understand the colonization ability of Shigella spp. To study colonization ability, an animal model of 4-day-old suckling mouse, was exploited. We characterized the HA activity of 48 Shigella strains, with erythrocytes collected from rabbit, guinea pig, chicken, and sheep. Only rabbit and guinea pig erythrocytes showed positive HA reactions in most of the cases. On the basis of HA pattern, 4 strains from each serogroup were selected for in vivo colonization studies. Our results showed a positive correlation between HA activity and colonization ability of the strains belonging to different serogroups (groups A, B, C, and D) of Shigella. In all 4 serogroups, high HA titer was associated with greater intestinal colonization.

Diazo Transfer and Click Chemistry in the Solid Phase Syntheses of Lysine-Based Glycodendrimers as Antagonists against Escherichia coli FimH

Uropathogenic Escherichia coli infections, ultimately leading to cystitis and pyelonephritis, are initially mediated by the adhesion of the bacterial FimH to the transmembrane glycoprotein uroplakin-1a present at the surface of urothelial cells. The adhesion is based on the recognition and high avidity binding between the high-mannose glycans of the uroplakin and the FimH, a mannose-specific lectin located at the tip of type 1 fimbriae. We found that synthetic multiantennary mannopyranosides glycodendrons, harboring triazole functionality at the anomeric position, were potent hemagglutination inhibitors of guinea pig erythrocytes and E. coli. A mannosylated dendrimer exposing up to sixteen sugar residues showed an HAI titer of 1 μM and was thus 500-fold more potent than the corresponding monovalent methyl α-d-mannopyranoside. The synthesis of the glycodendrons involved highly efficient solid-phase synthesis of branched l-lysine scaffolds, diazo transfer reaction on the terminal amine residues, and 1,3-dipolar copper-catalyzed azide-alkyne cycloaddition using propargyl α-d-mannopyranoside.

Comparative kinetics of organophosphates and oximes with erythrocyte, muscle and brain acetylcholinesterase

There is an ongoing debate whether oximes can effectively counteract the effects of organophosphorus compounds (OP) on brain acetylcholinesterase (AChE) activity and whether there are differences in the kinetic properties of brain and erythrocyte AChE. In order to investigate the kinetics of AChE from different tissues and species the well established dynamically working in vitro model with real-time determination of membrane-bound AChE activity was adapted for use with brain AChE. The enzyme reactor, that was loaded with brain, erythrocyte or muscle AChE, was continuously perfused with substrate and chromogen while AChE activity was on-line analyzed in a flow-through detector. It was possible to determine the Michaelis–Menten constants of human erythrocyte, muscle and brain AChE which were almost identical. In addition, the inhibition kinetics of sarin and paraoxon as well as the reactivation kinetics of obidoxime and HI 6 were determined with human, swine and guinea pig brain and erythrocyte AChE. It was found that the inhibition and reactivation kinetics of brain and erythrocyte AChE were highly comparable in all tested species. These data support the view that AChE from different tissue has similar kinetic properties and that brain AChE is comparably susceptible toward reactivation by oximes.

Low Hemagglutinin-Titer Strains of Pandemic Influenza A (H1N1) 2009 Virus Circulated in Toyama Prefecture, Japan, during the 2009–2011 Influenza Seasons

*Corresponding author: Mailing address: Department of Virology, Toyama Institute of Health, 17-1 Nakataikoyama, Imizu-shi, Toyama 939-0363, Japan. Tel:+81-76656-8143, Fax: +81-766-56-7326, E-mail: masatsugu. obuchi@pref.toyama.lg.jp Fig. 1. Comparison of the HA titers of pandemic influenza A (H1N1) 2009 virus isolates obtained during the 2009–2010 and 2010–2011 seasons in Toyama Prefecture, Japan. HA assay was performed with 0.75z guinea pig erythrocytes. Jpn. J. Infect. Dis., 64, 2011

Single nucleotide polypmorphisms of fimH associated with adherence and biofilm formation by serovars of Salmonella enterica

Type 1 fimbriae produced by serovars of Salmonella are characterized by their ability to agglutinate guinea pig erythrocytes in the absence of d-mannose but not in its presence. The FimH protein is the adhesin that mediates this reaction; it is distinct from the major fimbrial protei.n (FimA) that composes the fimbrial shaft. Avian-adapted serovars of Salmonella produce non-haemagglutinating fimbriae that have been reported to mediate adherence to avian cells. A single amino acid substitution is present in the FimH adhesin of these strains compared to that of a Typhimurium isolate. Also, previous studies have shown that single nucleotide polymorphisms in two strains of the Typhimurium fimH alter the binding specificity. We therefore investigated the allelic variation of fimH from a range of serotypes (both host-adapted and non-host-adapted) and isolates of Salmonella. Most FimH adhesins mediated the mannose-sensitive haemagglutination of guinea pig erythrocytes, but many did not facilitate adherence to HEp-2 cells. A small number of isolates also produced fimbriae but did not mediate adherence to either cell type. Transformants possessing cloned fimH genes exhibited a number of different substitutions within the predicted amino acid sequence of the FimH polypeptide. No identical FimH amino sequence was found between strains that adhere to erythrocytes and/or HEp-2 cells and those produced by non-adherent strains. FimH-mediated adherence to HEp-2 cells was invariably associated with the ability to form biofilms on mannosylated bovine serum albumin.

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.

Isolation and Characterization of a Novel Cardiac Stimulatory and Haemolytic Protein from the Sea Anemone Urticina piscivora (Sebens and Laakso)

Ethanolic extraction of the whole bodies of the sea anemone Urticina piscivora, collected from the west coast of Canada contained a protein UpI with potent cardiac stimulatory and haemolytic activities. The protein was purified by gel chromatography, cation exchange, preparative and analytical reverse phase HPLC and shown to have a molecular size of approximately 28 kDa and pI of >9·3. The partial amino acid sequence as determined by automated Edman degradation of whole UpI is D1E-NEN5LYGPN10ENKAK15AKDLT20A GASY25LTKEA30GCTKL35QAGCT40MYQAY45N. The sequence was not identical to any of the known cardiotonic polypeptides and proteins, or cytolysins, already characterized from sea anemones. However it showed some sequence similarity to the phospholipase A2-like bungarotoxins from the snake venom of Bungarus multicintus. Even though U. piscivora belongs to the family Actiniidae, UpI lacks the initial N-terminal residues common to sea anemone proteins from this family. Using rat left atrial tissues, UpI had an ED50 of 8·1 × 10−9 M. We have shown UpI to be a potent haemolysin on erythrocytes of rat, guinea-pig, dog, pig and human. Like most sea anemone cytolysins, the haemolytic action is inhibited in a dose dependent manner by the phosholipid sphingomyelin. It was also ichthyotoxic. We thus conclude, that UpI is a novel cardiac stimulant and cytolysin by virtue of its biological action, molecular size and biochemical data. We believe we have successfully characterized for the first time a member of the third class of sea anemone cytolysin described as having phospholipase A2-like action, but for which no biochemical data are yet available.