Characterization of naturally elaborated blebs from serum-susceptible and serum-resistant strains of Neisseria gonorrhoeae

Outer-membrane blebs from two serum-susceptible and two serum-resistant strains of Neisseria gonorrhoeae were characterized. In general, bleb surfaces resembled cell surfaces, but there were qualitative and quantitative protein differences in blebs released by serum-susceptible and serum-resistant strains. Relative to blebs from serum-resistant strains, blebs from serum-susceptible strains expressed reduced amounts of major outer-membrane proteins I and III, and little if any 68,000 Dalton outer-membrane protein.     

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.     

Comparative Electrophoretic and Amino Acid Analyses of Isolated Membranes from Streptococcus pyogenes and Stabilized L-Form

Major quantitative, but not qualitative, differences in the various species of proteins in purified membranes from Streptococcus pyogenes and its stabilized L-form have been demonstrated by acidic and alkaline disc gel electrophoresis with and without urea. The fact that no significant differences in the amino acid content or composition between these two membranes could be demonstrated emphasizes that these results are probably due to changes in the relative amounts of the various species of proteins in this subcellular component. The possibility of these protein changes in the L-form membrane being related to its inability to synthesize a rigid cell wall is discussed. Finally, phage-associated lysin, routinely used for removal of the group A streptococcal cell wall, does not appear to affect the protein profile or amino acid composition of the membrane either metabolically or nonmetabolically.     

Characterization of the adherence properties of human Lactobacilli strains to be used as vaginal probiotics

In the present work, the adhesion of 43 human lactobacilli isolates to mucin has been studied. The most adherent strains were selected, and their capacities to adhere to three epithelial cell lines were studied. All intestinal strains and one vaginal isolate adhered to HT-29 cells. The latter was the most adherent to Caco-2 cells, although two of the intestinal isolates were also highly adherent. Moreover, five of the eight strains strongly adhered to HeLa cells. The binding of an Actinomyces neuii clinical isolate to HeLa cells was enhanced by two of the lactobacilli and by their secreted proteins, while those of another two strains almost abolished it. None of the strains were able to interfere with the adhesion of Candida albicans to HeLa cells. The components of the extracellular proteome of all strains were identified by MALDI-TOF/MS. Among them, a collagen-binding A precursor and aggregation-promoting factor-like proteins are suggested to participate on adhesion to Caco-2 and HeLa cells, respectively. In this way, several proteins with LysM domains might explain the ability of some bacterial supernatants to block A.?neuii adhesion to HeLa cell cultures. Finally, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) could explain the good adhesion of some strains to mucin.     

Two-dimensional gel analysis of proteins in cell lines from the central nervous system of larvalDrosophila

Summary High resolution two-dimensional gel electrophoresis was used to quantitatively analyze the patterns of protein synthesis in three different clones of a nerve cell line (ML-DmBG2) ofDrosophila melanogaster. When patterns of pulse-labeled proteins of the three different clones were compared, I observed quantitative variations affecting the rate of synthesis by twofold or more in 25–30% of the polypeptides and qualitative differences, always affecting less than 2% of the polypeptides. Patterns of protein synthesis were analyzed during the 24 d of culture, revealing both quantitative (increase or decrease; 40%) and qualitative (presence or absence; 3%) differences. More than 70 proteins synthesized in these cultures were secreted into the medium. Among them were two major groups of acidic proteins which disappeared with culture time. When cell lines and intact central nervous systems were compared, large differences in protein synthesis were observed. In fact, only 20% of the synthesized proteins were common to both isolated cells grownin vitro and the original nervous systemin vivo.     

Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines.

An improved method of two-dimensional gel electrophoresis has been developed which produces high resolution, reproducible images suitable for computer analysis. In the images that are presented, more than 800 proteins have been resolved without significant overlap, and many more proteins can be detected after longer exposures. To establish the usefulness of such methods for detailed quantitative comparisons of cultured cells, extensive controls have been carried out to test the reproducibility of the electrophoretic procedures, the sample preparation procedures, and the cell culture conditions. A computerized scanning system has been developed which can automatically detect and integrate the densities of the spots on a two-dimensional fluorogram or autoradiogram. The corresponding proteins from two or more samples can then be matched and their intensities compared. Several types of graphic output have been used to display the number and magnitude of the differences between the compared samples. These methods were used to study the patterns of protein synthesis in the nerve cell line B103 and the glial cell line B9. Both exponentially dividing and stationary cultures were analyzed and the relative rates of synthesis of approximately 300 proteins were compared by computer. For each cell line, no major qualitative differences were found between dividing and stationary phase cells although numerous quantitative differences of up to 15-fold were detected. The proteins that were increased or decreased in rate of synthesis as B103 cells became confluent were in general not the same proteins that were increased or decreased in rate of synthesis as B9 cells reached confluence, indicating that most of the changes do not reflect growth control responses common to all cells. When the two cell lines were analyzed in the same state of growth and compared by computer, qualitative differences were found in approximately 5% of the proteins analyzed, and at least 40% of the shared proteins were involved in quantitative differences of 2-fold or more. The rates of synthesis of the shared proteins were more divergent between the two cell lines than between dividing and stationary phase cells of either line. These studies show, therefore, that these cell lines can be distinguished, regardless of growth state, by their cell-specific proteins and by their characteristic rates of synthesis of many of the shared proteins.     

Bound amino acids in local strains of Trichomonas vaginalis

Amino acid composition of water-soluble and water-insoluble proteins of 8 strains of Tr. vaginalis is studied. 17 amino acids are found in both protein hydrolyzates. Despite the complete coincidence of their qualitative compositions there are reliable differences in the quantitative contents of some amino acids. Differences in the contents of main amino acids of water-soluble proteins of different strains reflect the belonging of the latter to different sero-groups. No reliable differences in the quantitative contents of amino acids of both water-soluble and water-insoluble proteins in strains belonging to one sero-group are recognised.     

Comparative release of alkaline phosphatases from Serratia marcescens by osmotic shock and polymyxin B treatment.

The comparative release of periplasmic enzymes and proteins from two strains of Serratia marcescens by osmotic shock and polymyxin B treatment was studied. There were significant qualitative and quantitative differences in the materials released by these two techniques. The osmotic shock procedure released a higher level of alkaline phosphatase activity and a greater number of protein components than the polymyxin B treatment. The molecular weights of the active components released by the two techniques were shown to be 190,000 +/- 10,000 (A'), 140,000 +/- 10,000 (A) and 110,000 +/- 10,000 (B) daltons. Components released by polymyxin B were also released by osmotic shock. However, the reverse was not true. Component B in the osmotic shock fluids was by far the most active. The differences in the release mechanisms of the two techniques were discussed. It is suggested that polymyxin B treatment is the method of choice because of its selectiveness and mildness, despite the rather low level of activity of alkaline phosphatase released.     

Differential proteomic analysis of HeLa cells treated with Honokiol using a quantitative proteomic strategy

Honokiol (HNK) is an active component purified from Magnolia officinalis. HNK exhibits antitumor effects by inducing apoptosis and inhibiting the growth of many cancer cell lines, while proteins involved in antitumor effects in proteomic level are still unclear. In our study, HNK could inhibit HeLa cell proliferation and induce apoptosis in a concentration- and time-dependent manner. We utilized a quantitative proteomic technique termed SILAC (Stable isotope labeling with amino acids in cell culture)-MS (mass spectrometry) to study the differential proteomic profiling of HeLa cells treated by HNK. A total of 85 proteins were changed after HeLa cells were treated with 12 microg/ml HNK for 8 h, and 8 proteins showed up-regulation while 77 proteins down-regulated. The changed proteins were classified into 9 different categories, which covered a broad variety of cellular functions. In conclusion, HNK performs cytotoxicity to HeLa cells through co-operating of many proteins and different pathways.     

Surface proteins on Cloudman melanoma cells

Lactoperoxidase-catalyzed cell surface radioiodination was used to study the size distribution and physiological characteristics of cell surface proteins of Cloudman melanoma cells maintained in vivo (An cell) or in vitro (TC cells). Slight qualitative and quantitative differences were observed between detergent-extracted radiolabeled membrane proteins obtained from An and TC cells when the proteins were resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Striking differences were observed in the membrane residence times of radioiodinated proteins of the An and TC cells when labeled cells were cultured 24 hr. Many labeled surface proteins are lost more rapidly from An cells than from TC cells. The possibility that the release of surface proteins from tumor cell membranes may be an important factor which determines the nature of the host immune response against incipient cancers is considered.     

A Recombinant Measles Vaccine Virus Expressing Wild-Type Glycoproteins: Consequences for Viral Spread and Cell Tropism

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.     

Proteomic analysis of striated muscle

The techniques collectively known as proteomics are useful for characterizing the protein phenotype of a particular tissue or cell as well as quantitatively identifying differences in the levels of individual proteins following modulation of a tissue or cell. In the area of striated muscle research, proteomics has been a useful tool for identifying qualitative and quantitative changes in the striated muscle protein phenotype resulting from either disease or physiological modulation. Proteomics is useful for these investigations because many of the changes in the striated muscle phenotype resulting from either disease or changes in physiological state are qualitative and not quantitative changes. For example, modification of striated muscle proteins by phosphorylation and proteolytic cleavage are readily observed using proteomic technologies while these changes would not be identified using genomic technology. In this review, I will discuss the application of proteomic technology to striated muscle research, research designed to identify key protein changes that are either causal for or markers of a striated muscle disease or physiological condition.     

Schistosoma japonicum: disc electrophoretic protein patterns of the Japanese, Philippine, and Formosan strains

Soluble proteins of the Japanese, Philippine, and Formosan strains of Schistosoma japonicum were separated by disc electrophoresis using polyacrylamide gel columns. Differences between strains and sexes were investigated on the basis of R_f values of the bands, location of prominent peaks, quantitative comparisons of major bands, and overall densitometric profiles. With male extract, 29, 28, and 29, distinct bands were resolved for the Japanese, Philippine, and Formosan strains, respectively. Female extracts gave 31, 26, and 25 distinct bands for the Japanese, Philippine, and Formosan strains, respectively. Both qualitative and quantitative differences were found among the three strains and between sexes. The closest relationship of densitometric profile was between the Japanese and Formosan strains.     

Surface characters and extracellular toxins involved in the pathogenesis of Aeromonas hydrophila

A small number of serotypically distinct strains of A. hydrophila obtained from diseased freshwater fish were examined for their pathogenic properties comprising of cell surface characteristics and extracellular toxins. Test strains exhibited homogeneity in their cell surface characteristics despite being serologically heterogeneous. Studies on extracellular biological activities revealed qualitative and quantitative differences in production of toxins, probably explaining their antigenic diversity. Three distinct proteases, namely heat stable metallo protease, heat labile serine protease and heat labile metallo protease were identified from the strains.     

Induction of alkaline phosphatase and the glycoprotein hormone α subunit in HeLa cells by inhibitors of DNA polymerase

Levels of the glycoprotein hormone α subunit and alkaline phosphatase activity were increased in cultures of HeLa S₃ cells exposed to aphidicolin (0.2–10 μg/ml) or phosphonoformic acid (0.1–3 mm), inhibitors of DNA polymerase α. Induction was dependent on both the concentration and duration of exposure to the inhibitors and was prevented by cycloheximide and actinomycin D. Limited characterization of the induced α subunit and alkaline phosphatase activity suggest that they are similar to the uninduced proteins expressed by this cell line. Induction of both proteins by aphidicolin and phosphonoformic acid was enhanced by the simultaneous addition of 3 mm sodium butyrate but was depressed by 1 mm hydroxy urea. In contrast, both butyrate and hydroxy urea cause induction of these proteins when added alone to HeLa cultures. It is unlikely that a direct relationship exists between protein induction and the inhibition of DNA synthesis produced by aphidicolin and phosphonoformic acid since the concentrations required to produce half-maximal induction are 5 to 10 times greater than those needed to inhibit replication by 50%.     

Protein kinase activity and substrates at the surface of intact HeLa cells

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.     

Composition and genetic variability of heparin-sepharose CL-6B protein fractions obtained from the solubilized proteins of mouse organs

The solubilized proteins of liver and brain from mice of two inbred strains (C57BL/6J and DBA/2J) and their hybrids were subfractionated by heparin Sepharose (H-S) CL-6B affinity chromatography. The H-S binding and nonbinding proteins were separated by two-dimensional electrophoresis. The protein patterns obtained were analyzed with regard to their protein composition and their genetic variability (qualitative and quantitative variants). Eighty to ninety percent of the H-S binding proteins were unique to this class of proteins. This class was rich in organ-specific proteins. Compared to the nonbinding proteins the portion of basic proteins was only slightly increased, suggesting that most of the H-S binding proteins interact specifically with heparin. The frequency of qualitative protein variants revealed that H-S binding proteins are more conservative than H-S nonbinding proteins. The quantitative genetic variability was higher in liver than in brain. Quantitative protein variants occurred more frequently than qualitative variants.