Antigenic Behaviors of Two Axial Flagellar Proteins Detected in Treponema denticola

Two polypeptide antigens with molecular sizes of 34,000 daltons (34 kDa) and 38 kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34-kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38-kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A-gold complexes demonstrated that the 38-kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34-kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34-kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.     

Molecular Cloning of a Murine cDNA Encoding a Novel Protein, p38-2G4, Which Varies with the Cell Cycle

Proliferating cells express genes active in cell cycle control. The modulation of control genes and factors are required to maintain critical cell cycle activities. We used a set of monoclonal antibodies prepared against DNA-binding proteins from Ehrlich ascites tumor cells in immunofluorescent microscopy to screen for proteins showing cell cycle-specific staining patterns. Here, we report cloning and characterizing of a novel mitogen-inducible gene from murine macrophages that predicts a cell cycle-specifically modulated nuclear protein of 38 kDa, designated p38-2G4. p38-2G4 displayed a speckled pattern of varying fluorescence intensity confined to the nucleus, but sparing the nucleoli. Strongly stained granules were observed between G1 and mid S phase, followed by a less abundant punctated arrangement toward the end of S phase, and negative fluorescence at the S/G2 transition. Thereafter, the nuclear staining reappeared. Additionally, p38-2G4 expression vanished in G0-arrested cells and was restored after release from growth arrest. p38-2G4 conserved in vertebrates by means of immunofluorescence data contains a number of putative phosphorylation sites, a cryptic nuclear localization signal, and an amphipathic helical domain. Our cDNA and its deduced amino acid sequence is related to a Schizosaccharomyces pombe gene encoding a 42-kDa protein that associates with curved DNA, suggesting that we have cloned the murine homologue of the S. pombe gene which defines a novel cell cycle-specifically modified and proliferation-associated nuclear protein in mammals.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles

The yeast [PSI⁺] prion originates from the self-perpetuating transmissible aggregates of the translation termination factor Sup35p. We previously showed that infectious Sup35p particles are exported outside the cells via extracellular vesicles (EV). This finding suggested a function for EV in the vertical and horizontal transmission of yeast prions. Here we report a significant export of Sup35p within periplasmic vesicles (PV) upon glucose starvation. We show that PV are up to three orders of magnitude more abundant than EV. However, PV and EV are different in terms of size and protein content, and their export is oppositely regulated by glucose availability in the growth medium. Overall, our work suggests that the export of prion particles to both the periplasm and the extracellular space needs to be considered to address the physiological consequences of vesicle-mediated yeast prions trafficking.     

Tubulin isotypes in maize endosperm. Alterations during development and water deficit

Maize ( Zea mays L. cv . Pioneer 3925) endosperm development is sensitive to water deficit during rapid cell division and nuclear DNA endoreduplication. To gain insight into effects of water deficit on gene-products that are involved in these processes, we examined the accumulation of β-tubulin, a 50-kDa subunit of microtubules. Proteins extracted from endosperms were separated by SDS-PAGE and immunoblotted with antibodies to β-tubulin. In addition to the expected 50-kDa β-tubulin protein, monoclonal antibodies recognized a 35-kDa protein that predominated at early stages of development and progressively disappeared coincident with the appearance of 50-kDa β-tubulin. Various tests demonstrated that the cross-reacting 35-kDa protein was not a post-harvest artifact, but represented a group of in situ tubulin isotypes preferentially detected by the monoclonal antibodies we used. The pattern of appearance of the fragment suggested that differential expression or degradation of tubulin isotypes normally occurs during development. This expression pattern is prologed or altered during water deficit, which may affect cell division.     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Macromolecules Involved in the Amoeba-Bacteria Symbiosis1

ABSTRACT. In the Amoeba-bacteria symbiosis, rod-shaped Gram-negative bacterial endosymbionts reside within symbiosomes in the host cytoplasm, and the host and symbionts are mutually dependent for survival. Three proteins and one group of lipopolysaccharides (LPS) synthesized by the bacterial endosymbionts and two proteins derived from the host cells have been found to be involved in the host-symbiont interactions, although their respective roles are not yet fully known. The symbiont-derived molecules included proteins with molecular weights of 29 kDa, 67 kDa and 96 kDa and LPS. The 29-kDa protein was most abundant in the host cytoplasm, while the 96-kDa protein and LPS were found mostly on the symbiosome membranes. The 67-kDa protein was a GroEL analog and stayed within the symbionts. The host-derived 43-kDa protein, actin, was selectively accumulated by the symbionts, while the 220/225-kDa protein, spectrin, was attached to the symbiosome membranes. The symbiont genes coding for the 29-kDa and 67-kDa proteins were cloned and sequenced. The 29-kDa protein gene was unique with no relation to any known DNA sequences but has a leucine zipper-like motif, suggesting a possible DNA-binding function. The DNA sequence of the 67-kDa protein gene showed a 70% identity with heat-shock-protein genes of Escherichia coli and Coxiella burnetii.     

Coevolution of actin and associated proteins: an α-actinin-like protein in a cyanobacterium (Spirulina platensis)

Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells. Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria. Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e. Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins. We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins. We also report for the first time that α-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein. Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an α-actinin-like protein using specific antibodies.     

Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis

Abstract Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38–40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38–40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

This study elucidates the role of the protein kinases p37γ and p38δ in regulating the metabolic switch that occurs in early postnatal development, revealing that they inhibit glycogen synthase 1 and glycogen metabolism. Deregulation of this mechanism results in cardiac defects and metabolic alterations which can be prevented by maternal fatty acid diet supplementation during pregnancy and lactation.     

Cloning of a Partial Length cDNA Encoding the C-Terminal Portion of the 75-77-kDa Antigen of Trypanosoma cruzi

ABSTRACT It has been suggested that several Trypanosoma cruzi antigens have possible protective epitopes which may be suitable vaccine candidates. We found previously that animals resistant to T. cruzi infection produced antibodies against the 75-77-kDa parasite antigen. To test the ability of the recombinant form of this antigen to protect animals from T. cruzi infection, the cDNA which encodes a portion of the 75-77-kDa antigen was cloned using a cDNA library constructed in an orientation-specific bacteriophage expression vector (λgt11) from poly (A)⁺ RNA of Brazil strain epimastigotes. One clone, named SFS-40, was selected by screening the library using affinity purified antibodies specific for the 75-77-kDa parasite antigen as probe. The cDNA corresponding to the 1.7-kilobase insert of SFS-40 was subcloned into plasmid vectors and characterized. The cDNA sequence encodes a polypeptide of about 40 kDa. The putative product of the cDNA was homologous to members of the 70-kDa stress protein family. When epimastigotes were shifted from 29° C to 37° C, there was no change in the level of SFS-40 mRNA. In contrast, the 70-kDa heat shock protein mRNA of the parasite was increased about four fold by this treatment.     

Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions

The pollination droplet is a highly conservative pollination mechanism that is observed in all major gymnosperm taxa. Proteomics analysis of the pollination drops was carried out on four gymnosperm species: Juniperus communis (common juniper), Juniperus oxycedrus (prickly juniper), Chamaecyparis lawsoniana (Port Orford cedar), and Welwitschia mirabilis. Pollination drop proteins were purified by SDS-PAGE, and the most abundant proteins were analyzed by mass spectrometry and sequenced. Based on BLAST searching of combined amino acid sequences, the following proteins were identified in the following species: an 83-kDa subtilisin-like proteinase, a 62-kDa glycosyl hydrolase, a 47.5-kDa glucan 1,3-β-glucosidase precursor, a 30-kDa chitinase, and a 25-kDa thaumatin-like protein were identified in J. communis; a 30-kDa chitinase, a 25-kDa thaumatin-like protein, and a 32.5-kDa glucanase-like protein were identified in J. oxycedrus; an 83-kDa subtilisin-like proteinase, a 62-kDa β-d-glucan exohydrolase, a 47.5-kDa glucan 1,3-β-glucosidase, and two 25-kDa thaumatin-like proteins were identified in C. lawsoniana, and a 25-kDa chitinase was identified in W. mirabilis. Based on protein identifications, there is strong evidence that the pollination drop functions in both pathogen defense and pollen development. The discovery of similarities in terms of peptide sequence and protein identifications indicates that ovular secretions are functionally conservative, and that they are essential to reproductive success.     

Lipopolysaccharide core components of Rhizobium etli reacting with a panel of monoclonal antibodies

Monoclonal antibodies that react with Rhizobium leguminosarum lipopolysaccharide core antigens (LPS-2) have been used to investigate LPS-2 structure in Rhizobium etli. The panel of antibodies (JIM 32 - JIM 35, JIM 37, JIM 38) specific for LPS-2 of R. leguminosarum strain 3841 and its core components displays similar reactivities towards isolated LPS-2 from R. etli CE109 (a mutant of wild-type strain R. etli CE3 that displays LPS-2 as its main LPS form on the cell surface). This result suggests the antibodies bind to similar epitopes on both strains and, hence, that R. leguminosarum and R. etli have very similar LPS core and lipid A antigen structures. More detailed analysis of the antibody binding sites with isolated LPS-2 and lipid A from R. etli suggests that some of the antibodies (JIM 32, 33, 34, and MASM-I) bind some part of the core oligosaccharides, while others (JIM 35 and JIM 38) involve lipid A. These antibodies have already proven useful in the biochemical analysis of the LPS antigen forms. For example, the loss of reactivity of certain LPS forms with antibody JIM 37 has led to the discovery of a hitherto unnoticed form of the LPS antigen in a precipitate formed during the phenol/water extraction procedure. This new form reacts with the JIM 37 antibody. Furthermore, the positive reaction of some of the antibodies with only sonicated wild-type R. etli cells suggests that either an effective way of masking the display of core antigens on whole bacterial cells is occurring or that core forms of the LPSs are never displayed on the surface of the bacterial cells. Either possibility, once confirmed, could be important for our picture of the Rhizobium cell surface and could also have some bearing on symbiotic nodule infection and development.Abbreviations LPS lipopolysaccharide     

The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate,and fumarate by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1

Background  

Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.