Strong Hemolytic Activity Research Articles

HlyIA-like sequence present in a genetic locus with low homology to appA in Actinobacillus pleuropneumoniae serotype 10.

Using the appA gene encoding Actinobacillus pleuropneumoniae-RTX-toxin II (ApxII) as a probe, Southern hybridization demonstrated the presence of appA-sequence in all 12 serotypes except serotype 10. Serotype 10 strains had a genetic locus with low homology to appA (tentatively designated Lha). DNA sequence analysis revealed that Lha contained a part of hlyIA gene encoding a 105-kDa hemolysin ApxI. Southern hybridization demonstrated the presence of Lha (hlyIA) sequence not only in serotype 10 but also in serotypes 1, 5a, 5b, 9, and 11, which had strong hemolytic activity with sheep red blood cells and were highly virulent for mice. These results suggest that ApxI may be associated with the virulence phenotype of the organism.

Haemolysin‐derived synthetic peptides with pore‐forming and haemolytic activity

Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990). A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.

Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies

Monoclonal antibodies (MAbs) were raised against extracellular hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae serotypes 2 and 9. MAbs were tested for inhibition of hemolytic and cytotoxic activities of the reference strains of A. pleuropneumoniae serotypes 1 to 12. Five MAbs inhibited hemolytic or cytotoxic activities of one or more serotypes and were used to probe Western blots (immunoblots) of cytotoxic and hemolytic culture filtrates of serotypes 1 to 12. The MAbs recognized three different proteins: (i) a protein of approximately 103 kDa that was associated with a weak hemolytic activity and a moderate cytotoxic activity, (ii) a protein of approximately 105 kDa that was associated with a strong hemolytic activity and a strong cytotoxic activity, and (iii) a protein of approximately 120 kDa that was associated with a strong cytotoxic activity but not with hemolytic activity. Serotypes 6, 7, and 12 produced only the 103-kDa protein, and serotype 10 produced only the 105-kDa protein. The other serotypes produced two proteins: serotypes 1, 5, 9, and 11 produced the 103- and 105-kDa proteins, and serotypes 2, 3, 4, and 8 produced the 103- and 120-kDa proteins.

Properties of a deep Proteus R mutant isolated from clinical material

Some biological features of a deep P. mirabilis 17301 R mutant isolated from the urine of a patient with chronic UTI were studied and compared with similar features of P. mirabilis S forms and five induced Proteus R mutants of different chemotypes. There were no differences in lethal toxicity and adhesion to human uroepithelial cells. Of all the R mutants tested, two of them, 17301 and R4, exhibited strong cell-bound hemolytic activity. The P. mirabilis R 17301 was characterized as the most invasive (tested in L929 mouse fibroblasts) compared to the other Proteus S and R forms. The structure of PS from a clinical R mutant investigated and the results of serological studies prove that this mutant belongs to the Rc chemotype.

Triterpenoidsapogenine in der GattungGrindelia

A saponin fraction has been isolated from the ethanolic extract of Grindeliae herba. The saponin fraction showed strong haemolytic activity. The saponins were detected after separation on TLC by their color reactions and Rf values as well as by their haemolytic activity on blood agar. After hydrolysis, the chemical reactions and Rf values of the sapogenins were described. The main sapogenin was isolated by preparative chromatography and its structure was elucidated by mass, 1H-NMR, 13C-NMR, IR, and UV spectra. The compound is a new natural triterpenoid sapogenin lactone and is named grindeliasapogenin D. Spectral evidence is presented to establish its structure as 2 beta,3 beta,16 alpha,23-tetrahydroxyoleanan-28,13 beta-olide. Two further sapogenins present in small amounts were identified chromatographically as oleanolic acid and bayogenin using authentic reference substances. The aerial parts of Grindelia robusta Nutt, and G. lanceolata Nutt. (Asteraceae) (as well as two commercial extracts) were also investigated with respect to their saponin fraction. The 3 sapogenins mentioned above were detected in all samples. The antibiotic potency of the crude drug is not associated with the saponin fraction but is at least partially due to the resin fraction.

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain)

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.

Hemolysis Mechanism of Dental Adhesive Monomer (Methacryloyloxydecyl Dihydrogen Phosphate) Using a Phosphatidylcholine Liposome System as a Model for Biomembranes

To clarify the mechanism of interaction of dental adhesive monomers with biological membranes at the molecular level, we studied the interaction of methacryloyloxydecyl dihydrogen phosphate (MDP) and methacrylic acid (MAA) with the dipalmitoylphosphatidylcholine (DPPC) liposome system using NMR and DSC. MDP-DPPC interaction became apparent through broadening of the DPPC phase transition as pH decreased, finally the enthalpy of MDP-DPPC (1:1 mol ratio) reduced to zero at pH 2.5. Proton chemical shifts of MDP enhanced shielding and proton signals due to the phosphatidylcholine polar group (O-CH2-CH2-N bond) of DPPC were observed. MAA-DPPC interaction was smaller than that of MDP-DPPC, even at low pH. It was concluded that the strong hemolytic activity of MDP may be due to its interaction with the phospholipid bilayers of erythrocyte membranes.

There are two C4 genetic loci and a null allele in the chimpanzee.

Genetic polymorphism in C4 in the chimpanzee was studied by agarose gel electrophoresis of desialated plasma and development of patterns by immunofixation with antiserum to human C4 and by a C4-sensitive hemolytic overlay. In general, immunofixation patterns showed multiple partially overlapping bands of which only the most cathodal had strong hemolytic activity. In analogy to human C4, the latter were designated C4B, whereas those detected by immunofixation which had little hemolytic activity were designated C4A. Chimp C4A and C4B reacted with human and mouse (monoclonal) anti-C4B and human anti-Ch1 but neither reacted with monoclonal anti-C4A or human anti-Ch2, Ch3, Rg1, or Rg2. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the alpha chain of C4B showed a slightly lower apparent relative mass than that of C4A at around Mr 93,000. There were three C4A variants and two C4B variants inherited in families as autosomal codominant traits, as C4A-C4B cosegregating pairs with no detectable crossing-over. These pairs were inherited with chimpanzee leukocyte antigen types C2 and BF variants without detectable crossing-over. Half-null C4 haplotypes with C4B QO were observed in family studies. Nine BF, C2, C4A, C4B allelic haplotypic combinations (complotypes) were identified among presumably unrelated chimpanzees.

Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization.

The cytoplasmic, dense granules of cloned T cell lines were isolated and analyzed for their functional and biochemical properties. Isolated granules of approximately 90% homogeneity, in the presence of Ca, effect strong tumoricidal and hemolytic activity. Tumor cell lysis is complete in less than 30 min, with less than 2 micrograms granule protein corresponding to a killer/target ratio of 3-6:1 by assuming 50% yield for granule isolation. The granules contain a set of unique proteins, responsible for cytolytic activity and designated K1 to K6, in the molecular weight range of 14,000 to 75,000, as defined by sodium dodecyl sulfate (SDS) polyacrylamide slab gel analysis under reducing and nonreducing conditions. Cytolysis mediated by isolated granules is accompanied by the assembly of tubular complexes of 160 A (poly P1) and of approximately 70 A width (poly P2) that are inserted into membranes and form ultrastructural membrane lesions. As shown by immunofluorescence and by Percoll gradient fractionation, cytolytic granules are detected in cells of cytolytic T cell lineage and not in the T cell lymphomas E14 and S194. Poly perforin 1 assembled by CTLL-2 upon stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA) was isolated by detergent extraction and gel filtration. Poly P1 is composed of disulfide-linked subunits that, after reduction, co-migrate with certain granule proteins. The results are compatible with the hypothesis that the dense granules of cytolytic T cells contain cytolytic proteins that polymerize to disulfide-linked tubular poly perforins in a Ca-dependent reaction and may cause cytolysis by membrane insertion and transmembrane channel formation.

Isolation and characterization of the third component of complement in the serum of the clawed frog, Xenopus laevis.

The hemolytic activity against SRBC in the serum of normal Xenopus is dependent on specific antibody and both Ca++ and Mg++, whereas the activity against RRBC is dependent on Mg++ alone. Both of these hemolytic activities disappeared after treatment of the serum with zymosan or with the specific rabbit antiserum against one of the zymosan-binding proteins in Xenopus serum. By using this antiserum as a probe, a complement component (XC) was purified as a single entity from the Xenopus plasma after polyethylene glycol precipitation, DEAE-Sepharose CL-6B, Sepharose CL-6B, and Sephadex G-200 column chromatographies. The XC, contained at 2.3 mg/ml in normal serum, showed an electrophoretic mobility of beta-globulin, with a m.w. of 204,000 (204K) comprising two distinct subunits of 125K and 85K, which are linked with each other by disulfide bonds. The 204K protein exhibited a strong hemolytic activity in association with other components in Xenopus serum. Digestion of 204K protein by trypsin resulted in a specific cleavage of the 125K subunit and a conversion of its immunoelectrophoretic mobility to the anodal side, leaving the 85K subunits intact. The treatment of XC with SDS and urea resulted in the splitting of 125K subunits into 78K and 40K, but this splitting was inhibited upon pretreatment with methylamine, suggesting the presence of a thiol ester bond in the XC. The amino acid composition of the XC revealed a striking resemblance to that of mammalian C3. In all aspects, the 204K protein (XC) is regarded as representing the C3 of Xenopus laevis, which plays a key role in both the classical and alternative hemolytic pathways.

The hemolytic activity of chrysotile asbestos fibers: A freeze-fracture study

Short asbestos fibers isolated by a sedimentation procedure have a strong hemolytic activity. In the presence of ferritin particles, hemolysis by chrysotile fibers is inhibited at least during the first 10 min. Freeze-fracture studies show that after 20 sec or 2 min of contact between the fibers and the RBC membrane, the intramembranous particles remain randomly distributed over the whole surface of the P-face. On the E-face of the asbestos-treated red blood cell membranes, the number of intramembranous particles is significantly reduced. With the transmission electron microscopy, it is not possible to resolve the trilaminar structure of the ghost membrane around the deeply buried asbestos fibers. It is postulated that the membrane defects brought about by asbestos are caused by the adsorption of one membrane constituent, possibly phospholipids, on the chrysotile fibers.

Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis.

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.

Nature of Melittin-Phospholipid Interaction

Melittin, the major protein component of bee venom that possesses a strong hemolytic activity (1), is an amphipathic protein whose amino acid residues 1-20 are predominantly hydrophobic; residues 21-26 are hydrophilic. Determination of the molecular basis and specificity of the attractive forces involved in its interaction with membranes is essential for elucidating the mode of its biological action. In the present work we have employed the nanosecond and steady-state fluorometric properties of the single tryptophan residue of the protein (located at position 19) for probing the degree of penetration of that residue into phospholipid liposomes and the state of aggregation of the protein when bound to the liposomes.

Nonoccluded virus-like particles in the mollusc Agriolimax reticulatus (Stylommatophora: Limacinae)

Mass mortality of ark clam, Scapharca broughtonii, occurred in a local farm along the Bohai Sea, China in August 2017. Ostreid herpesvirus SB strain, which was the only pathogen previously reported in S. broughtonii, was not detected in moribund S. broughtonii. However, four kinds of bacteria were isolated from the hepatopancreas of naturally diseased S. broughtonii, and they were further identified based on 16S rDNA gene sequence analysis. These bacteria were closely related to Vibrio harveyi (A2-1), Microbulbifer variabilis (A2-2), V. alginolyticus (A2-3), and Pseudoalteromonas sp. (A2-4). Hemolysis test showed that A2-1 exhibited a strong hemolytic activity. The cumulative mortality of S. broughtonii with foot-injected A2-1 reached 100%, which was significantly higher than that of groups infected with other bacteria. Notably, temperature showed a significant effect on the mortality of S. broughtonii infected with A2-1, as the immersion challenged test showed 100% mortality occurred at 25 ± 2 °C on the 9th day, whereas no death was observed at 15 ± 2 °C on the 12th day. Temperature also significantly affected the growth rate and extracellular protein secretion of A2-1. In conclusion, this study is the first to report that V. harveyi is a pathogen that causes mass mortality in S. broughtonii, and the temperature is an important environmental factor that affects the virulence of A2–1 on S. broughtonii.

CELL LYSIS AND CHANGES OF SHAPE OF ERYTHROCYTES BY INJECTIONS

Cell lysis and changes of shape were examined to elucidate the effects of injections on rat erythrocytes. Hemolysis did not occur within the range of pH 5.5-8.0 and 160-700 mOsm/kg in a period of 2 hrs at 37°C. Clear change of shape were not observed within the range of pH 7.0-8.0 and 280-300 mOsm/kg in a period of 2 hrs at 37°C by a scanning electron microstopy. Benzyl alcohol, ethyl alcohol and propylene glycol showed strong hemolytic activity at the concentration, regularly contained in injections. Benzyl alcohol produced internalization of erythrocytes ; however clear morphological change was not observed with ethyl alcohol or propylene glycol. The effects of other drugs or ingredients in injections were also examined.

Further studies on the lethal effect of long-chain fatty acids on mycobacteria.

Mycobactericidal activity of long-chain fatty acids was confirmed by in vitro exposure of BCG. The killing effect was accompanied by inhibition of the membrane-bound acid phosphatase activity. Such active fatty acids were those having a stronger hemolytic activity (e.g., C12:0, C14:0, C18:1, C18:2). Heat-killed BCG cells or their cell walls adsorbed the toxic fatty acids, whereas the fatty acid-insensitive E. coli cells did not. It was suggested that the mycobactericidal action of long-chain fatty acids is due to their detergent-like action on the cytoplasmic membrane, and that the determinant factor for the fatty acid-sensitivitiy of bacteria is the property of the cell wall by which fatty acids are adsorbed so that the active site is brought into contact with the inner membrane.

Toxicity of the cardiotoxic protein, flammutoxin, isolated from the edible mushroom Flammunlina velutipes

Flammutoxin is a cardiotoxic protein which has been purified from the edible mushroom, Flammulina velutipes. The protein has strong hemolytic activity against human group O red blood cells and calcium ions at physiological concentration can partially reverse the hemolytic effect. The inhibitory effect of the toxic protein on the respiration of Ehrlich ascites tumor cells was also suppressed by calcium ions. When the toxic protein was injected i.p. into mice, it caused the writhing reaction which was partially inhibited by antihistaminics. When the toxic protein was locally injected into rat paws, it induced severe edema.

Identity of Streptolysin-O and NAD-Glycohydrolase (EC 3.2.2.5)

The homogeneity of purified NAD-glycohydrolase (EC 3.2.2.5) from Group C streptococci was demonstrated by analytical ultracentrifugation, disc electrophoresis and immunochemical analysis. Together with earlier characterization procedures the data show that the enzyme is homogenous in molecular seize, molecular form and electrophoretic charge. In addition, the enzyme has been shown to possess strong hemolytic activity which could be activated by thiol compounds. It was concluded from the analytical data that NAD-glycohydrolase- and Streptolysin-O-activities are catalytic functions of one protein molecule. Therefore the lysis of red blood cells by Streptolysin-O may be regarded as an enzymatic process catalyzed by an NAD-glycohydrolase.

Hemolytic factors of vibrio parahemolyticus. 1. Hemolytic activity of the supernatant fluid of its culture

It is well known that most strains of Vibrio parahaemolyticus originated from fuman patients demonstrate on a specially prepared blood agar plate a hemolytic activity called Kanagawa-phenomenon, whereas those from sea-fish and sea-water fail to show such an activity. In this paper, a hemolytic activity of the supernatant of the broth culture was investigated and, at the same time, its relationship with Kanagawaphenomenon was studied.Strongly Kanagawa-phenomenon positive strains were inoculated into brain heart infusion broth and incubated in 37°C water bath with rocking culture method.Supernatant fluid of the broth culture showed strong hemolytic activity against human red cells and lethal toxicity to mice, but, no effect to horse red cells. These activities were resistant to heating at 100°C for 30 min. but inactivated by tripsin. The production of the cytolytic factor was observed in the period of logarithmic multiplication of the vibrio.On the other hand, another hemolytic factor which was revealed thermolabile and resistant to tripsin was recognized in the lysate of Vibrio cells not only from Kanagawa-phenomenon positive but also negative cultures. This hemolytic factor lysed both human and horse red cells but demonstrated no toxicity to mice.

Phosphonolipids. XVII. Synthesis of ethylene glycolphosphonic acid analogues of lecithins.

The synthesis of ethylene glycolphosphonic acid analogues of lecithins, a new type of pbosphonolipids, is reported. The preparation of myristoyl, palmitoyl, and stearoyl ethylene glycol-(2-trimethylammoniumethyl)phosphonate is described in detail. They were obtained by phosphonylating the corresponding esters of ethylene glycol with (2-bromoethyl)phosphonic acid monochloride and triethylamine, and treating the resulting acyl glycol-(2-bromoethyl)phosphonates in dimethylformamide with trimethylamine at 50–55° for 3 days. The glycolphosphonolecithins (phosphonic acid analogues of glycollecithin) are highly soluble in water and possess strong hemolytic activity. On hydrolysis by lecithinase C (Clostridium welchii) or alkali (Dawson procedure) they yield the corresponding monoacyl glycols and ethylene glycol-(2-trimethylammoniumethyl)phosphonate, respectively. The identification of ethylene glycolphosphonolecithins in the presence of phosphonolecithins, glycollecithins, and lecithins by descending paper chromatography of their saponification products, obtained by mild alkaline hydrolysis, is described.