In vitro hemolytic activity ofPlasmodium berghei on red blood cells

A suspension ofPlasmodium berghei obtained by lysis with saponin of red blood cells from an infected rat showed high hemolytic activity, when incubatedin vitro with normal rat red blood cells. The hemolysis was a temperature-dependent process and was dependent on the concentration of the parasite. Plasma ofPlasmodium berghei infected albino rats also possessed lytic activity.     

Phospholipase activity and virulence of pathogenic leptospirae

Results of investigation of phospholipase activity and virulence of pathogenic leptospirae on a solid nutrient medium using the method of agar layers with 1% of L-lecithin in the medium of the second layer are presented. It has been demonstrated that only one zone of translucent medium is formed around the colonies of pathological leptospirae, which is obviously due to the release of phospholipase A. The examined virulent strains of pathogenic leptospirae were found to exhibit greater phospholipase activity (width of the translucent zones being 5.0 +/- 0.34 mm) than the avirulent strains (width of the zones being 1.5 +/- 0.11 mm).     

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract ofP. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37 ℃, and at pH 7.0; Hg2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract of P. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37℃, and at pH 7.0; Hg^2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg^2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Synthesis and biological evaluation of backbone-aminated analogues of gramicidin S

We report the parallel synthesis of gramicidin S derivatives featuring backbone N-amino substituents. Analogues were prepared by incorporation of N-amino dipeptide subunits on solid support. Nine backbone-aminated macrocycles were evaluated for growth inhibitory activity against ESKAPE pathogens and hemolytic activity against human red blood cells. Diamination of the Orn residues in the β-strand region of gramicidin S was found to enhance broad-spectrum antimicrobial activity without a corresponding increase in hemolytic activity.     

Purification of a hemolytic factor from ras oncogene transformed fibroblasts

Transformation of NIH-3T3 fibroblasts by the Harvey murine sarcoma viral oncogene or by cultivation of fibroblasts under low serum conditions (spontaneous) resulted in the acquisition of hemolytic activity, as demonstrated by coincubation of the transformed fibroblasts with 59Fe-labeled red blood cells. The tumor Hemolytic Factor was partially purified from conditioned media produced by T-24 human bladder transformed fibroblasts by ammonium sulfate precipitation, followed by anion and gel filtration chromatography. The hemolytic factor has a molecular weight of 66,000 as documented by SDS-PAGE and is destroyed by heating to 60 degrees C.     

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.     

Comparison of the efficacy of different techniques, culture media, and sources of blood in determining the hemolytic activity of Listeria spp.

Hemolytic activity is a fundamental criterion for the differentiation of Listeria species; therefore, a simple and inexpensive procedure to clearly distinguish hemolytic strains from each other and from nonhemolytic strains would be of great aid. We compared the efficacy of several techniques, culture media, and types of blood in demonstrating the hemolysis of Listeria spp. The hemolytic activities of Listeria monocytogenes and Listeria seeligeri were more easily detected with a red blood cell top-layer (RBCTL) technique and with a microplate technique than when the strains were streaked on blood agar (BA). Listeria ivanovii produced a marked hemolysis regardless of the technique employed. In general, the hemolytic activity of these three species was stronger on media containing brain heart infusion (BHI) agar and (or) potassium tellurite (PT). However, Listeria innocua produced questionable hemolytic reactions when nonselective culture media with BHI and PT were utilized, limiting the advantages gained by employing the two compounds. The RBCTL and the BA techniques disclosed greater hemolytic activity for L. monocytogenes, L. seeligeri, and L. ivanovii with sheep and guinea pig blood than with horse and human blood. When the microplate technique was used, all four kinds of blood were equally effective.     

Hemolytic activity of Vibrio cholerae eltor and V. cholerae O139 toxigenic and nontoxigenic strains

A total of 20 ctx- and 16 ctx+ V. cholerae eltor strains, 20 ctx- and 22 ctx+ V. cholerae O139 strains were under study. Hemolytic activity was tested in modified Greig test with sheep, guinea pig and rabbit red blood cells. The comparative study of the hemolytic properties of V. cholerae O1 and O139 under different conditions of cultivation demonstrated their capacity of lysing sheep red blood cells (SRBC) irrespective of the presence of toxigenic properties. A wider spectrum of lytic activity of ctx- strains in Greig test with respect to red blood cells of different animals and the capacity of lysing SRBC, most resistant to the action of toxin, may be due to a considerably greater content of Hly+ clones in their population.     

Effect of Statolon-induced Interferon on Antibody Formation

The effect of statolon-induced interferon on immune response was investigated. Young adult Swiss albino mice were immunized with chicken red blood cells or infectious mengovirus. The red blood cells were injected three times and the virus twice at 2-week intervals, with or without treatment with statolon 24 hr before the inoculations. The effect of interferon on antibody formation was measured by determinations of hemagglutinating, hemolytic, and virus-neutralizing antibodies in the sera of treated and nontreated groups. The hemagglutinin and hemolysin titers after each successive inoculation were equal in both groups, indicating that interferon had no effect on their production. A decrease in the primary response to the viral antigen was associated with interferon induction; the booster response was unaffected. The possible use of interferon inducers as attenuants for immunization with live virulent viruses is discussed.     

Synthesis and biological activity of novel macrocyclic antifungals. modification of the tyrosine moiety of the lipopeptidolactone FR901469.

A series of tyrosine-modified derivatives of the macrocyclic lipopeptidolactone FR901469 have been prepared and evaluated for in vitro and in vivo antifungal activity and for hemolytic activity towards red blood cells. Compound 14 displayed significantly reduced hemolytic potential at 1mg/mL and a comparable protective effect to FR901469 in a mouse candidiasis model.     

A hemolytic protein secreted from epidermal cells of the Arabian Gulf catfish, Arius thalassinus (Ruppell)

1. A fright or shock induced toxic secretion (gel) from the epidermis of the Arabian Gulf catfish, Arius thalassinus, exhibits hemolytic activity when tested against red blood cells from many different sources. 2. An enzyme with hemolytic activity, which represents 1.1% of the total soluble gel protein fraction, has been purified to homogeneity. 3. Molecular sieve chromatography and SDS polyacrylamide gel electrophoresis of the purified protein indicate a mol. wt of 34,000. 4. One additional protein component with hemolytic activity was found in the epidermal secretion. 5. Specific activity of the catfish epidermal factor is 20.6 units/mg protein, a level somewhat lower than those of most protein hemolytic factors. 6. The catfish hemolytic factor was not ichthyotoxic when tested against small fish and did not cause lethality when administered intravenously to rabbits.     

Hemolytic Activity of a Togavirus,Getah*

A purified toga-alphavirus, Getah (GET), showed optimal hemolytic activity for one-day-old chick red blood cells when incubated at 37 C for 120 min at pH 6.2. Experimental data obtained from various angles, such as pH dependency, inhibition by virus-specific antiserum and by concanavalin A, indicated that the hemolysis was a property of the virus particle itself. Although the mechanism of hemolysis by togaviruses has not been known, our results indicated that viral lipids may participate in this activity since the hemolytic activity was impaired by delipidation procedures.     

Death and Lysis of Leptospirae When Cultured in Asbestos-Filtered Growth Media

Death and lysis of leptospirae, when cultured in asbestos-filtered bovine albumin polysorbate 80 media, was quantitated. The pathogens (virulent and avirulent) required 2 x 10(6) cells/ml to initiate growth in such media, whereas inocula of 2 to 20 cells/ml grew in control medium. Saprophytic leptospirae initiated growth from 2 cells/ml in asbestos-filtered medium as well as control medium. The adverse action of asbestos-filtered medium was not removed by storage of medium for 2 years at 25 C and was not diminished when such medium was frozen at -80 C. Washing with water, HCl and NaHCO(3)-NaCl, citric acid, and medium components did not remove the lytic activity associated with asbestos-filtered culture medium. Continuous subculture in asbestos-filtered medium was possible from large inocula; however, upon subsequent dilution and reinoculation into asbestos-filtered media, there was no evidence of acquired resistance, and all pathogens failed to grow.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

Characteristics of a new abnormal variant of G-6-PD in human red cells.

Kinetic and electrophoretic properties were studied in 230--300 fold purified preparations of glucose-6-phosphate dehydrogenase (G-6-PD) from red cells of donors and patients with hemolytic anemia induced by G-6-PD deficiency. In abnormal variant of G-6-PD isolated from red cells of a patient with hemolytic anemia which had not before been described in the literature was found. The abnormal variant differs from the normal enzyme by a decreased Michaelis constant for G-6-P and NADP, by increased utilization of substrate-analogues (2-deoxy-G-6-P and deamino NADP in particular), by low heat stability, the character of pH dependence, and by the appearance of one band of G-6-PD activity during electrophoresis in polyacrylamide gel. The isolated abnormal variant of G-6-PD has been called "Kremenchug" according to the origin of the patient.     

Identification of alternative pathway serum complement activity in the blood of the American alligator (Alligator mississippiensis)

Incubation of different dilutions of alligator serum with sheep red blood cells (SRBCs) that had not been sensitized with antibodies resulted in concentration-dependent hemolytic activity. This hemolytic activity was not affected by the presence of ammonium hydroxide and methylamine, known inactivators of the classical complement cascade. However, the hemolytic activities were inhibited by EDTA and salicylaldoxime, indicating that the alternate pathway is primarily responsible for these activities. Immunofixation of electrophoretically-resolved alligator serum proteins with antihuman C3 polyclonal antibodies resulted in detection of a protein antigenically similar to human C3 in alligator serum. SDS-PAGE, followed by Western blot analysis, revealed the presence of two alligator serum proteins with nearly identical molecular weights as human C3alpha and C3beta. SRBC hemolysis and antibacterial activity by alligator serum was significantly reduced in the presence of antihuman C3 antibodies. The hemolytic effect of alligator serum was shown to occur rapidly, with significant activity within 5 min and maximal activity occurring at 15 min. SRBC hemolysis was also temperature-dependent, with reduced activity below 15 degrees C and above 30 degrees C. These data suggest that the antibiotic properties of alligator serum are partially due to the presence of a complement-facilitated humoral immune response analogous to that described in mammalian systems.     

Detection of a new anomalous variant of glucose-6-phosphate dehydrogenase in human erythrocytes]

Kinetic and electrophoretic properties of 230--300 fold purified preparations of glucose-6-phosphate dehydrogenase (G6PD) from red cells of donors and patients with acute drug hemolytic anemia due to G6PD deficiency were studied. A new abnormal variant of G6PD isolated from red cell of a patient with acute drug hemolytic anemia, which was not described in literature, has been discovered. The abnormal enzyme differs from the normal by decreased Michaelis constant for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP), by increased utilization of analogues of substrates--2-deoxy-glucose-6-phosphate and particularly deamino-NADP, by low thermal stability, by the character of pH-dependence, by the appearance of a single band of G6PD activity in polyacrylamide gel electrophoresis.     

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.