Red cell membrane glycophorin labeling from within the lipid bilayer.

Human red blood cell membranes were labeled from within the lipid bilayer by the apolar photosensitive reagent, 5-[125I]iodonaphthyl-1-azide. Glycophorin, the major sialoglycoprotein of the red cell membrane, was purified by two different methods; it contained approximately half of the total label incorporated into membrane proteins. The label was confined to the trypsin-insoluble peptide of glycophorin that includes a sequence of 20, mainly apolar, amino acids. These findings provide direct evidence that the labeled segment resides within the membrane in direct contact with the lipid bilayer, and support the suggestion that glycophorin spans the bilayer through its hydrophobic domain.

Receptor-mediated internalization of fluorescent chemotactic peptide by human neutrophils.

Tetramethylrhodamine labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys is a potent chemoattractant for human neutrophils. Binding of this peptide to living neutrophils was observed by means of video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization of the fluorescent peptide. These processes are dependent on specific binding to the formal peptide chemotactic receptor.

Role of superoxide anion in host cell injury induced by mycoplasma pneumoniae infection. A study in normal and trisomy 21 cells.

The role of Mycoplasma pneumoniae-generated superoxide and hydrogen peroxide in inducing host cell injury was studied in normal and trisomy 21 human cells. As a result of M. pneumoniae infection, catalase activity in infected normal skin fibroblasts and ciliated epithelial cells decreased by 74-77% as compared with uninfected controls. Addition of superoxide dismutase to the infected cultured cells totally prevented the inhibition whereas addition of catalase or catalytically inactivated superoxide dismutase had no protective effect. Trisomy 21 erythrocytes and cultured skin fibroblasts in which CuZn-superoxide dismutase content is 50% greater than in normal cells were infected by M. pneumoniae. The inhibition of catalase activity in these cells was 7-33% and 0-20.5%, respectively, as compared with 65-72% and 48-68% inhibition in normal infected controls. Following M. pneumoniae infection, the levels of malonyldialdehyde, an indicator for membrane lipid peroxidation were raised in trisomy 21 cultured fibroblasts by 10-32% while in normal cells malonyldialdehyde increased by 140-870%. Externally added superoxide dismutase, but not catalase, reduced the extent of lipid peroxidation in normal infected cells. Lactate dehydrogenase release from normal infected cells was time correlated with the increase in their malonyldialdehyde formation. It is suggested that superoxide generated during M. pneumoniae infection is involved in the inhibition of host cell catalase activity. The inactivation of this cellular antioxidative defense mechanism results in progressive oxidative damage to the M. pneumoniae-infected cells.

Stimulation of lymphoid cells by components of BCG.

BCG was fractionated into a delipidated mycobacterial cell fraction (DMC) and lipid by exhaustive chloroform-methanol extraction. The effects of these fractions were tested on mouse spleen cells, nonadherent spleen cells (lymphocytes), thymus cells, and adherent spleen cells (macrophages) in vitro and were compared with effects of the whole bacilli and a methanol-extraction residue (MER). Tritiated thymidine incorporation into spleen cells, purified spleen lymphocytes, and thymus cells was measured as an indicator of activity on these cells; lymphocyte-activating factor (LAF) production was used to measure activation of macrophages. DMC and MER were at least equivalent to, and often exceeded, whole BCG in their stimulation of spleen cells and spleen lymphocytes. DMC was a poor thymic mitogen in contrast to MER, which was as strong as BCG in this regard. Lipid was far less effective a mitogen for all cells tested, and failed to augment the effectiveness of DMC on thymus cells when both were present in the incubation mixture. LAF production was significantly increased by whole BCG (18-fold above controls), whereas each fraction increased production threefold to sixfold. These in vitro results seemed to reflect the known in vivo activity of BCG and its components and suggest further antitumor applications.

Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes.

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3-5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures.

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.

Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents.

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.

Characterization of monoclonal antibodies to human platelet myosin that recognize highly conserved epitopes within the 50 kDa fragment of myosin subfragment-1.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.

The erythrocyte membranes in beta-thalassemia. Lower sialic acid levels in glycophorin.

The sialic acids content of glycophorin of thalassemic erythrocyte membranes is about 25% lower than in glycophorin of normal erythrocyte membranes. Glycophorin extracted from old thalassemic erythrocytes separated by density centrifugation, has about half the sialic acids content found in glycophorin extracted from young thalassemic erythrocytes. Possible sialidase activty was sought in the plasma and erythrocyte membranes of thalassemic erythrocytes. No increased sialidase activity was detected in the plasma of the patients as compared to that of normal donors. Thus, other sites for sialidase activity, or other possibilities have to be explored to account for the increased sialic acid hydrolysis of glycophorin of the thalassemic erythrocytes.

Preliminary X-ray diffraction results on co-crystals of wheat germ agglutinin with a sialoglycopeptide from the red cell receptor glycophorin A.

Diffraction-quality crystals have been obtained for complexes of each of the major wheat germ agglutinin (WGA) isolectins with the tryptic sialoglycopeptide T-5 from the WGA red cell receptor glycophorin A. This octa-glycopeptide possesses a Thr-linked carbohydrate moiety (GalNAc(NeuNAc)-Gal-NeuNAc) with specificity for the WGA binding site. The crystals belong to the orthorhombic space group P2(1)2(1)2 and have unit cell dimensions: a = 112.2 A, b = 51.0 A, c = 63.5 A (isolectin 1); a = 109.0 A, b = 52.3 A, c = 62.4 A (isolectin 2). There are two monomer complexes in each asymmetric unit.

Mycoplasmas regulate HIV-LTR-dependent gene expression.

Mycoplasmas have been incriminated in setting the stage for HIV infection and full-blown AIDS. We tested the possible involvement of mycoplasmas in activation of HIV. Two cell lines, 293 fibroblasts and Jurkat CD4+ T-cells, transfected with plasmids harboring a transcription fusion construct between HIV-long terminal repeat (HIV-LTR) and either luc or cat genes, were infected with several mycoplasmas (M. fermentans; M. penetrans, M. pirum and Ureaplasma urealyticum) and the reporter gene expression was monitored. The data presented here suggest that mycoplasmas, and specifically their membranes, play a role in the activation of HIV-LTR mediated transcription.

Adherence of Mycoplasma pneumoniae to human alveolar macrophages.

The human pathogen Mycoplasma pneumoniac causes primary atypical-cold agglutinin-positive pneumonia. Since alveolar macrophages internalize mycoplasma as part of their immune defense, we studied characteristics of the human macrophage receptor for opsonized and nonopsonized M. pneumoniae. The glass-adhering subpopulation of M. pneumoniae attached more than the non-adherent subpopulation. The attachment was dose-dependent and enhanced by opsonization in the presence of human serum. It is inhibited by sulfated compounds such as dextran-sulfate and polyanetholsulfonic acid, but not by dextran or several monosaccharides, suggesting that sulfated glycolipids on the macrophage surface may act as receptors for M. pneumoniae binding. In addition, sialylated compounds, such as fetuin and alpha 1-acid glycoprotein, were found to be potent inhibitors of the attachment, also indicating the role of sialic acid residue in recognition and attachment of M. pneumoniae to human alveolar macrophages.

The effect of antibodies to human platelet membrane and cytoplasmic myosins on myosin ATPase activity and platelet aggregation.

Myosins were purified from the membrane fraction and the cytoplasm of human platelets. Polyclonal antibodies to the purified myosins were induced in rabbits. Their effects on the ATPase activity of the purified myosins as well as on the process of platelet aggregation were studied. A strong cross reactivity was found between the two myosins and their respective antibodies by the ELISA technique. It was found that the antibodies preferentially bind to the "head" segment of the myosins, since purified myosin "rod" reacted only weakly with the two kinds of antibodies. The two antimyosin antibodies strongly inhibited the K+(EDTA) ATPase activity of both myosins, as well as the activity of the isolated myosin "heads". The amount of antimembrane myosin antibody required to inhibit the above enzymatic activity was smaller than that of the anticytoplasmatic myosin antibody. Similar results were observed with F(ab)2 fragments of the two kinds of antibodies. No effect of these antibodies or their F(ab)2 fragments was observed on platelet aggregation induced by various agonists, although their inhibitory effect on the platelet myosin ATPase activity was strong.

Distribution of actin, myosin and actin binding protein in platelets of patients with hyperlipoproteinemia.

Significant anomalies in the quantity and relative distribution of the contractile proteins actin, myosin and actin binding protein (ABP) were observed in platelets obtained from patients with hyperbetalipoproteinemia (type IIa) and in patients with hypertriglyceridemia (type IV). Changes were observed in unfractionated platelets, (increased ABP in type IIa patients and increased actin in both type IIa and type IV) in isolated platelet membranes (increased ABP and actin in type IV, and increased myosin in type IIa) and in the KCl extract of platelets (increased actin in type IV and increased myosin in type IIa). The myosin ATPase specific activity was increased in platelets of type IV patients. No changes were observed in the concentrations and distribution of membrane glycoproteins in the platelets of these patients. The above anomalies in the contractile proteins might be relevant to the known functional anomalies of the platelets of patients with hyperlipoproteinemias.

Structural and immunological properties of myosin from human platelet external and internal membranes.

Our previous studies indicate that platelets contain two myosin isoforms, one of them localized in the membrane while the other in the cytoplasmic compartment. Structural and functional differences of these myosins have been characterized. In this study two platelet membrane subfractions, the external and the internal membranes, were isolated simultaneously from a crude membrane fraction and their purity was characterized using specific marker enzymes. Myosin was shown to be present in both membrane fractions and its structural and immunological properties were investigated. The electrophoretic mobilities of myosin in both membrane preparations were identical to the mobility of its cytoplasmic counterpart. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the maps of the myosins from the external and internal membranes as compared to their soluble counterpart. Our data suggest that myosin is located in three distinct platelet compartments: cytosol, external and internal membranes. The same myosin isoform is located in the two membrane compartments, while the isoform found in the cytosol is different. The observed variations in the structure of the two isoforms may reflect differences in their respective physiological functions.

Altered structural and functional properties of myosins, from platelets of idiopathic scoliosis patients.

Platelets of patients with idiopathic scoliosis (IS) have been shown to have decreased capacity to aggregate and secrete in response to certain agonists. Similarities between the contractile protein system of platelets and muscle have made the platelets a popular model for muscle disease. We attempted to characterize the function and structure of myosin in platelets of IS patients. Blood was obtained from seven IS patients and seven matched non-scoliotic healthy controls. The mean Cobb angle measurement of the IS patients was 35.4 degrees with a mean Risser sign of 2.2. Washed platelets were isolated from the blood, and the contractile proteins from the membrane and the cytosol compartments were isolated and analyzed by two-dimensional peptide mapping. As previously reported (J Biol Chem 258:9290, 1983), peptide maps of normal platelets revealed that the heavy chain of myosin located in the platelet membrane lacks one major spot relative to the cytoplasmic myosin. In IS patients the cytoplasmic myosin lacks the same peptide that is missing in the membrane myosin of normal individuals. In addition, the ATPase specific activity of the cytoplasmic myosin from IS platelets was significantly lower compared with the activity of the cytoplasmic myosin from normal platelets. These results suggest the presence of a fundamental abnormality of IS platelet contractile proteins.

Dual energy metabolism-dependent effect of Ureaplasma urealyticum infection on sperm activity.

Genital Ureaplasma urealyticum infection is considered a sexually transmitted infection. It has long been debated whether the presence of U. urealyticum in semen may be a possible cause of infertility. Long-term incubation (4 hours or overnight) of sperm cells with U. urealyticum in vitro resulted in a significant inhibition of sperm motility and membrane alteration whereas a short incubation (45 minutes) of sperm cells with ureaplasmas resulted in an acceleration of sperm velocity. The aim of this study was to understand these contradictory reports of U. urealyticum infection on sperm motility. Spermatozoa from fresh ejaculates of normozoospermic semen of men who were referred to the university Male Fertility Laboratory for semen analysis, with no history of genital tract infection, and from normal Assaf breed rams were infected in vitro with U. urealyticum serotype 8, at different pHs and O2 concentrations. Sperm viability and motility and changes in extracellular pH were evaluated. A significant (16%-43%) increase in sperm activity was observed upon infection at alkaline pH (7.8) under aerobic or hypoxic conditions, and a 58% increase was observed under anaerobic conditions and pH 7.2. When the infection was conducted under aerobic conditions and acidic pH (6.3), or under hypoxic conditions at neutral pH (7.2), an 8%-25% inhibition of sperm activity was observed. These results indicate that when sperm activity depends on mitochondrial oxidative phosphorylation, usually at low pHs, U. urealyticum competes with mitochondrial energy production and therefore reduces sperm motility and viability. However, when sperm energy metabolism depends on glycolysis, usually at higher pHs, U. urealyticum stimulates glycolysis and sperm activity.

"Lysosomal" enzyme activities in red blood cells of normal individuals and patients with homozygous beta-thalassaemia.

Four hydrolases, beta-galactosidase, beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase were examined in red blood cells (RBC) of normal donors and patients with homozygous beta-thalassaemia. Highly sensitive fluorimetric substrates were used to determine the specific activities of these enzymes. In order to avoid contamination by lysosomal activities derived from white blood cells (WBC), the mature RBV were separated from other blood elements by cellulose chromatography. The hydrolase activities in normal RBC were detected only in their plasma membranes and were found to be considerably lower than in WBC or platelets. In thalassaemic RBC, hydrolase activities were present in both plasma membranes and in the soluble fraction. The normoblast fraction contributed most of the hydrolase activity found in these preparations, suggesting the presence of lysosomal particles in thalassaemic RBC. No differences in the enzymatic activities were found when purified membranes of mature RBC from thalassemic and normal preparations were compared. The origin and roles of these hydrolytic enzymes in normal and thalassaemic RBC membranes are not known.

Caenorhabditis elegans: Stage Specific Differences in Cuticle Surface Carbohydrates.

Stage-specific differences in wheat germ agglutinin (WGA) binding saccharides were demonstrated between the surfaces of the eggs, L1 larvae, young aduhs, and old adults of Caenorhabditis elegans. The WGA binding was to n-acetylglucosamine groups but not to terminally linked n-acetylneuraminic acids. An age-related decrease in WGA binding occurred in adults, supporting previous findings of a decrease in net negative cuticle surface charge during aging.

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici.

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.