Lysosomal breakdown of erythrocytes in the sheep placenta

Summary The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (=erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.     

Final stages of erythrophagocytosis in the sheep placenta

Summary In trophoblastic epithelial cells of the sheep placenta the final stages of erythrocyte breakdown within the lysosomal apparatus were studied at the ultrastructural level.As a result of hemoglobin digestion lysosomes containing hemoglobin-derived pigments (HDP) were formed. The HDP-lysosomes were acid phosphatase-positive, highly electron-dense bodies of round to irregular shape containing whorled membranous formations. The accumulation of these lysosomes in epithelial cells led to fusion resulting in the formation of conglomerates. At the end of the gestation period the amount of HD Plysosomes and their conglomerates markedly increased.In addition to erythrocytes the trophoblastic epithelial cells in the erythrophagocytic regions phagocytosed maternal leukocytes and neighbouring epithelial cells and giant cells.By gradual accumulation of HDP-lysosomes and remnants of phagocytosed cells, highly electron-dense acid phosphatase-positive residual bodies of variable appearance were formed within the epithelial cells.At the end of pregnancy the spaces between juxtaposed villi of the trophoblastic epithelium in the erythrophagocytic zones were occluded by apposition of the epithelial cells. In these occluded regions an increase in highly electron-dense large-sized residual bodies (15–22 m of dimension) occurred as a result of multiple cell phagocytosis in combination with fusion. In these residual bodies the numerous incorporated HDP-lysosomes and the remnants of phagocytosed cells could still be recognized.     

Clostridium perfringens alpha-toxin activates the sphingomyelin metabolism system in sheep erythrocytes

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.     

Abnormalities of elastic and transporting properties of red blood cells under development of apoptosis

The functional properties of erythrocytes under development of apoptotic process in these cells were investigated by the low angle light scattering technique. Apoptosis induced by ionomycin was associated with an initial decrease of cell volume and caused formation of echinocytes. After that the cells restored their volume forming rounded erythrocytes with rugged membrane capable to agglomerate with each other. At the late stages of apoptosis, small fragmented cells can be revealed. Preapoptotic red blood cells (at all stages of apoptosis) manifested an enormous tolerance to hypotonic loading, whereas control cells hemolyzed just after reaching a critical volume (∼150 fl). Acidic hemolysis cannot differentiate between control and preapoptotic erythrocytes, the cells being hemolyzed not reaching the critical volume. Placing the control erythrocytes to a medium with ammonia ions instead of sodium ions caused an initial increase of cell volume above the critical point, and then it was also followed by hemolysis. Under ammonia loading, an initial rate of the cell volume growth and a ratio of the hemolyzed cells were significantly reduced in preapoptotic cells.     

Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.     

Investigations into combined dietary deficiencies of copper,selenium, and vitamin E in the rat

Interactions between dietary Cu, Se, and vitamin E in ascorbate-induced hemolysis of erythrocytes obtained from rats fed diets deficient or adequate in these elements were investigated. Hemolysis was affected by all three dietary factors, through closely interrelated but distinct mechanisms. In vitamin E-deficient cells, hemolysis was increased and the amount of hemolysis was directly related to the amount of hemoglobin breakdown. Deficiency of Cu or Se decreased hemolysis, but only in vitamin E-deficient cells. Vitamin E did not affect the breakdown of hemoglobin, but Cu and Se did. Hemolysis and hemoglobin breakdown were decreased by the addition of glucose, through mechanisms independent of that involving reduced glutathione metabolism. These results suggest that vitamin E acts within erythrocyte membranes to prevent products of hemoglobin breakdown from initiating peroxidation and subsequent hemolysis. Effects of Cu and Se are linked with that of vitamin E by the involvement of glutathione peroxidase and Cu superoxide dismutase in the cytoplasmic breakdown of hemoglobin, rather than by a direct effect of these enzymes on lipid peroxidation. It is concluded that the erythrocyte, because of its high heme content, probably represents a special system in terms of peroxidative pathways, and these findings may not be directly applicable to other tissues.     

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens alpha-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.     

Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium

Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event.     

Hemolysis induced by Bacillus cereus sphingomyelinase

Bacillus cereus sphingomyelinase (Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.     

Protease-nicked theta-toxin of Clostridium perfringens, a new membrane probe with no cytolytic effect, reveals two classes of cholesterol as toxin-binding sites on sheep erythrocytes

A nicked theta-toxin (C theta), obtained by limited proteolysis with subtilisin Carlsberg, causes almost no hemolysis while it retains a nearly intact cholesterol binding site below 20 degrees C. Neither electron microscopic evidence for the formation of arc- and ring-shaped structures on the membrane nor toxin-stimulated influx of extracellular Ca2+ are detected in C theta-treated cells below 20 degrees C. Thus, event(s) in the lytic process are responsible for the temperature dependency of hemolysis, which is also supported by the observation that C theta requires higher Arrhenius activation energy for hemolysis than the native toxin. Using C theta as a probe due to its high affinity for membrane cholesterol without causing any obvious membrane changes, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes. Both binding sites disappear by simultaneous treatment of the cells with sublytic doses of digitonin. Furthermore, C theta binds only to cholesterol among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes but not to the protein components derived from them. These results strongly suggest that cholesterol is an essential component of the both high- and low-affinity sites, and also imply that the modes of existence of cholesterol in the red cell membrane are heterogeneous.     

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O⁺ erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.     

Hemolysis in several animal species after rapid freezing of blood

The effect of various freezing rates on the extent of hemolysis in human, bovine and ovine erythrocytes, which are known to have different cell volumes, water contents and permeabilities, was investigated. Blood in stainless steel capillary tubes was frozen at various rates by abrupt immersion of the capillaries into cooling baths at temperatures ranging from ?20° to ?130°C. Minimum lysis values were obtained at freezing temperatures of ?40°, ?50° and ?70°C with, respectively, human, bovine and ovine blood. The smallest, highly permeable sheep erythrocytes were the least damaged at the highest freezing rates; the largest human cells with the highest water content, suffered the greatest damage; intermediate values were obtained with ox blood. At the lower freezing rates, the largest, human cells were the least damaged; the highest hemolysis values were obtained with the smallest, highly permeable sheep erythrocytes; ox blood again gave intermediate values. These results are in agreement with current views that, (1) very rapid freezing results in the formation of damaging intracellular ice; (2) injury associated with slow freezing is related to the extent of dehydration or to the increase in electrolyte concentration which accompanies ice formation; (3) minimum hemolysis is obtained under those freezing conditions in which osmotic dehydration has been sufficient to prevent the formation of intracellular ice, but has left enough water in the cells to prevent the damaging effects of dehydration and high electrolyte concentrations.     

Immunohistochemical localization of prostaglandin G/H synthase 1 and 2 in sheep placenta after glucocorticoid-induced and spontaneous labour

Enhanced prostaglandin production and release by the placenta is an essential element in the normal transition to labour in many animal species. In sheep, expression of prostaglandin G/H synthase (PGHS) is the central enzyme regulating this process. In this study immunohistochemistry was used to examine the distribution of cells expressing PGHS-1 and PGHS-2 in ovine placenta in association with spontaneous parturition (n = 6) and glucocorticoid-induced labour (n = 5). Labour was induced in ewes after the intrafetal injection of betamethasone on day 131 of gestation. Animals administered an intrafetal injection of isotonic saline (n = 5) acted as non-labour controls. In placentomes collected from all groups, immunoreactive PGHS-1 was present in the mononuclear trophoblast cells of the fetal placenta. Cells in the maternal mesenchyme and epithelial syncytium were weakly immunopositive for this enzyme. PGHS-1 immunoreactivity was also demonstrated in the endothelial cells of the chorionic vessels. The PGHS-2 isozyme was localized exclusively to the trophoblast epithelial cells. Immunoreactive PGHS-2 was not detectable in the maternal epithelial syncytium or the stroma of the cotyledons. The binucleate cells of the fetal placenta were consistently immunonegative for both PGHS isozymes. These results indicate that the cellular localization of PGHS-1 and PGHS-2 in ovine placenta does not change during the last 15 days of pregnancy. Co-localization of these isozymes indicates that the source of arachidonic acid and the site of prostanoid formation are the same. Quantitation of the percentage area of positive staining for PGHS-1 and PGHS-2 using image analysis software demonstrated a significant increase in PGHS-2 in the fetal trophoblast after glucocorticoid-induced labour and spontaneous parturition. This finding indicates that increased formation of the PGHS-2 isozyme is responsible for the large increase in prostaglandin production by the ovine placenta at term labour.     

Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai

It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis. We have isolated an Escherichia coli clone carrying the 7.8kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L. borgpetersenii. The clone showed a clear beta-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays. The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively. The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases. Interestingly, however, E. coli cells harboring sphH did not show sphingomyelinase or phospholipase activities. Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane. The SphH was successfully expressed in E. coli as a histidine (His)-SphH fusion protein. Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L. biflexa serovar patoc. We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.     

Similarities between complement-mediated and streptolysin S-mediated hemolysis

The oxygen-stable hemolysin streptolysin S (SLS) of Streptococcus pyogenes is encoded in part by the pel/sagA gene product. Antibodies to a synthetic peptide from the C terminus of the Pel/SagA open reading frame inhibited hemolysis mediated by both culture supernatants from multiple M serotypes of S. pyogenes isolates or a commercially available SLS preparation. Analysis of the SLS-mediated hemolytic reaction demonstrated that it was temperature- and concentration-dependent. Like complement-mediated hemolysis it conforms to the prediction of a one-hit mechanism of hemolysis. A number of intermediates in the SLS-mediated hemolysis of sheep erythrocytes could be distinguished. SLS could bind to erythrocytes below 17 degrees C; however, lysis could only occur at temperatures >23 degrees C. Following binding of SLS and washing, a papain-sensitive intermediate could be distinguished prior to insertion of the SLS complex into the erythrocyte membrane, which resulted in formation of a transmembrane pore and led to irreversible osmotic lysis of the cell. These intermediates were similar to those described previously during complement-mediated hemolysis.     

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.     

Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by "4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca²⁺-dependent while lysis is delayed in the absence of exogenous Ca²⁺. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.Abbreviations TPCK Tos-Phe-CH₂Cl,1-chloro-4 phenyl-3-L-tosylamidobutan-2-one - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - FCS Fetal Calf Serum     

Membrane fusion without cytoplasmic fusion (hemi-fusion) in erythrocytes that are subjected to electrical breakdown

There are many reports of hemi-fusion in phospholipid vesicles but few published studies on hemi-fusion in cells. We report evidence from both fluorescence microscopy and freeze-fracture electron microscopy for hemi-fusion in the electrofusion of human erythrocytes. We have also characterised the conditions that favour hemi-fusion as opposed to complete fusion, and discuss the possibility that hemi-fusion might precede complete electrically-induced cell fusion. A membrane probe (DiIC16) and a cytoplasmic probe (6-carboxyfluorescein) were used to investigate the behaviour of doubly-labelled human erythrocytes which were aligned in chains by dielectrophoresis and then exposed to high voltage breakdown pulses. Some of the cells were fused by the pulses, as shown by diffusion of both membrane and cytoplasmic probes from labelled to unlabelled cells. With other cells, the membrane probe diffused into unlabelled cells after the breakdown pulses, without the cytoplasmic probe diffusing into unlabelled cells or leaking into the medium. Membrane fusion (hemi-fusion) thus occurred without cytoplasmic fusion in these erythrocytes. Such cells were irreversibly, but fragilely, attached to their neighbours by the breakdown pulses. There was an inverse relationship between conditions that permit complete fusion and those that favour hemi-fusion, with respect to breakdown pulse length, breakdown voltage and, in particular, osmolarity and temperature. The incidence of hemi-fusion in 250 mM erythritol was twice that in 150 mM erythritol, and hemi-fusion was 5-fold greater at 25 degrees C than at 20 degrees C. Hemi-fused erythrocytes occasionally fused completely on heating to 50 degrees C, demonstrating that hemi-fusion can proceed to complete cell fusion. Freeze fracture electron micrographs of preparations of hemi-fused cells revealed long-lived, complementary depressions and protrusions on the E- and P-fracture faces, respectively, of tightly apposed cells that may mediate hemi-fusion. The possibility that the fusion of closely adjacent human erythrocytes by electrical breakdown pulses may involve an intermediate, shared bilayer structure, which is stable in certain conditions but which can be ruptured by osmotic swelling of the permeabilised cells, is discussed.     

Bacteriocin (hemolysin) of Streptococcus zymogenes

The sensitivity of Streptococcus faecalis (ATTC 8043) to S. zymogenes X-14 bacteriocin depends greatly on its physiological age. Sensitivity decreases from the mid-log phase on and is completely lost in the stationary phase. The sensitivity of erythrocytes to the hemolytic capacity of the bacteriocin showed considerable species variation. The order of increasing sensitivity was goose < sheep < dog < horse < human < rabbit. However, when red cell stromata were used as inhibitors of hemolysis in a standard system employing rabbit erythrocytes the order of increasing effectiveness was sheep < rabbit < human < horse < goose. When rabbit cells were used in varying concentrations with a constant hemolysin concentration, there was a lag of about 30 min, which for a given hemolysin preparation was constant for all red cell concentrations. Furthermore, the rate of hemolysis increased with increasing red cell concentration. If red cells are held constant and lysin varied, the time to reach half-maximal lysis varies directly with lysin but is not strictly proportional. Bacterial membranes were one to three orders of magnitude more effective than red cell stromata as inhibitors. The order of increasing effectiveness seems to be Escherichia coli < Bacillus megaterium < S. faecalis < Micrococcus lysodeikticus. In addition to membranes, a d-alanine containing glycerol teichoic acid, trypsin in high concentration, and deoxyribonuclease also inhibited hemolysis. Ribonuclease, d-alanine, l-alanine, dl-alanyl-dl-alanine, N-acetyl-d-alanine, N-acetyl-l-alanine did not inhibit hemolysis.     

Adhesive proteins of haemagglutinating Staphylococcus aureus isolated from bovine mastitis

Two proteins derived from the cell wall of Staphylococcus aureus, exhibiting apparent molecular masses of 116 kDa and 145 kDa, were found to bind to human buccal and bovine lactiferous sinus epithelial cells. By using antibodies specific for fibronectin-binding protein of S. aureus of human origin, the 116 kDa protein, but not the 145 kDa protein, was identified as a fibronectin-binding protein. The 145 kDa protein bound to bovine fat globule membranes, human buccal epithelial cells, bovine lactiferous sinus epithelial cells and sheep erythrocytes. The properties of the 145 kDa protein suggest that it is an adhesin with a possible role in the early stages of the development of bovine mastitis.