Characterization of tubular basement membrane antigens in human kidney

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.     

A self T cell epitope induces autoantibody response: mechanism for production of antibodies to diverse glomerular basement membrane antigens

The anti-glomerular basement membrane (GBM) Ab has been regarded as a prototypical example of pathogenic autoantibodies. However, the mechanism for elicitation of this Ab remains unknown. In the present paper, we report that the Ab to diverse GBM Ags was induced by a single nephritogenic T cell epitope in a rat model. The T cell epitope pCol(28-40) of noncollagen domain 1 of collagen type IV alpha3 chain not only uniformly induced severe glomerulonephritis but also elicited anti-GBM Ab in 76% of the immunized rats after prominent glomerular injury. Furthermore, we demonstrated that the anti-GBM Ab was not related to the peptidic B cell epitope nested in pCol(28-40); that is, 1) elimination of the B cell epitope, either by substitution of the critical residues of the B cell epitope or by truncation, failed to abrogate anti-GBM Ab production, and 2) the anti-GBM Ab, eluted from the diseased kidneys, reacted only with native GBM, but not with pCol(28-40). Confocal microscopy and immunoprecipitation further demonstrated that the eluted anti-GBM Ab recognized conformational B cell epitope(s) of multiple native GBM proteins. We conclude that autoantibody response to diverse native GBM Ags was induced by a single nephritogenic T cell epitope. Thus, anti-GBM Ab may actually be a consequence of T cell-mediated glomerulonephritis.     

Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.     

Demonstration of species-specific and cross reactive components of Paragonimus westermani crude worm antigen by EITB

Enzyme-linked immunoelectrotransfer blot (EITB) using crude worm antigen of adult Paragonimus westermani was performed for human patients sera to identify the species-specific components. Crude antigen was obtained by homogenizing and centrifuging 24-week old adult worms at 10,000 rpm for 60 minutes in phosphate buffered saline (PBS, pH 7.2) containing phenyl methyl sulfonyl fluoride (PMSF). Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and blotted electrophoretically onto a sheet of nitrocellulose paper. The sheet was cut into strips and exposed to sera diluted 1: 200 with PBS. SDS-PAGE showed 26 protein bands ranging 229 to 10 kDa. Of them 229, 91, 60, 50, 35-31, 27, 25, 21, 17, 11 and 10 kDa components showed positive reaction with serum antibody of patients with P. westermani. Sera of patients infected with Clonorchis sinensis reacted with 35-31, 19, and 11 kDa bands. Human sera from cysticercosis and diphyllobothriasis cases showed non-specific cross reactions with 229, 35-31, 27, 25 and 17 kDa bands. Protein bands of 91, 60, 21 and 10 kDa showed strong positive reaction without cross reactions with sera from other helminthic infections.     

Identification of human sperm surface glycoproteins recognized by autoantisera from immune infertile men, women, and vasectomized men

To identify the surface antigens of human sperm recognized by antisera from immune infertility patients and vasectomized men, we labeled sperm surface proteins with 125I- and used patient antisera for immunoprecipitation. Sera were studied from 27 infertile males, 18 infertile females, and 4 vasectomized males, each possessing anti-sperm antibodies detected by immunobead binding. Sera from different infertile males, different infertile females, and vasectomized males were remarkably similar in their surface antigen recognition. The different sera specifically immunoprecipitated the same small group of 125I-labeled surface proteins, which included polypeptides in the region 90 kDa, 40-45 kDa, and 26 kDa. Treatment with N-glycanase showed that the proteins of 90 kDa, 40-45 kDa, and 26 kDa were glycoproteins with N-linked carbohydrate. The immunoprecipitated 125I-labeled proteins and the total extract of 125I-labeled surface proteins were compared on two-dimensional (2D) gels. The results show the 90 kDa polypeptide is a major sperm surface component, whereas 40-45 kDa and 26 kDa polypeptides are minor components. The 2D gel comparison also indicates that 90 kDa, 40-45 kDa, and 26 kDa are a small subset of the total ensemble of sperm surface proteins. Clinical data suggest antibodies to these few proteins interfere with sperm function.     

Glomerular basement membrane. Identification of dimeric subunits of the noncollagenous domain (hexamer) of collagen IV and the Goodpasture antigen

The noncollagenous (NC1) domain hexamer of glomerular basement membrane (GBM) collagen is composed of a multiplicity of monomeric and dimeric subunits, and specific subunits are the targets for anti-GBM autoantibodies of patients with Goodpasture (GP) syndrome. The identity of GBM monomers has been established and the alpha 3(IV)NC1 monomer identified as the one that binds GP antibodies (Gunwar, S., Saus, J., Noelken, M. E., and Hudson, B. G. (1990) J. Biol. Chem. 265, 5466-5469). In the present study, the chain origin of 25 dimeric components and the identity of those that bound the anti-GBM antibodies from two GP patients were determined. This was accomplished by NH2-terminal sequence analysis and immunoblotting analysis of dimeric components that were resolved by two-dimensional electrophoresis in combination with high pressure liquid chromatography. The results revealed that (a) the components are mainly homodimers of the NC1 domains of alpha 1, alpha 2, alpha 3, alpha 4, and probably alpha 5 chains of collagen IV, reflecting a specificity of promoter-promoter association and (b) each homodimer had several size and charge isoforms. The GP antibodies bound exclusively to both alpha 3(IV)NC1 monomers and dimers and not to other basement membrane constituents. These findings provided new insights about the structure of GBM collagen and together with our previous findings firmly established the alpha 3(IV) chain as the target for the anti-GBM antibodies that mediate glomerulonephritis and pulmonary hemorrhage in patients with Goodpasture syndrome.     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

T cell epitope mimicry in antiglomerular basement membrane disease

Antiglomerular basement membrane (GBM) disease or Goodpasture's syndrome is among the earliest recognized human autoimmune diseases. Although collagen 4alpha3 NC1 (Col4alpha3NC1) has been identified as the responsible autoantigen, it remains unknown how autoimmunity to this autoantigen is provoked. We have demonstrated in our rat model that a single nephritogenic T cell epitope pCol28-40 of Col4alpha3NC1 induces glomerulonephritis. We hypothesized that microbial peptides that mimic this T cell epitope could induce the disease. Based on the critical residue motif (xxtTxNPsxx) of pCol28-40, seven peptides derived from human infection-related microbes were chosen through GenBank search and synthesized. All peptides showed cross-reactivity with pCol28-40-specific T cells at various levels. Only four peptides induced transient proteinuria and minor glomerular injury. However, the other three peptides induced severe proteinuria and modest to severe glomerulonephritis in 16-25% of the immunized rats. Unexpectedly, the most nephritogenic peptide, pCB, derived from Clostridium botulinum, also induced modest (25%) to severe (25%) pulmonary hemorrhage, another important feature of anti-GBM disease; this was not correlated with the severity of glomerulonephritis. This finding suggests that subtle variations in T cell epitope specificity may lead to different clinical manifestations of anti-GBM disease. In summary, our study raises the possibility that a single T cell epitope mimicry by microbial Ag may be sufficient to induce the anti-GBM disease.     

Mammalian FKBP-25 and its associated proteins

Soluble proteins from porcine brain were divided into two packs: (1) proteins which pass freely through CM52-cellulose, and (2) proteins retained on CM52. Each of these two packs of proteins was fractionated on preparative flat-bed isoelectrofocusing gel in the range of pH 2-12. Native FKBP-25 and its truncated forms were found among other proteins retained on CM52-cellulose. Immunoblotting with anti-FKBP-25 showed two bands in the range 27-30 kDa, one due to unmodified FKBP-25 and other due to FKBP-25 mixed with high-mobility group II protein (HMG-II). Selective immunostaining with anti-FKBP-25 antibodies of proteins which were not retained on CM52-cellulose showed several bands within the range of pI 7-5 and mass of 23 +/- 2 kDa. These fractions of proteins were next resolved on two-dimensional gels and immunostained with anti-FKBP-25 antibodies. Six proteins in the pI range 7-5 were detected. Edman degradation of alpha-chymotrypsin digests of the major spot suggests that it contains the GTP-binding protein Rab5 co-migrating with guanylyl kinase, whereas MALDI-TOF showed that a residual content of FKBP-25 may be also associated with these two proteins. A residual quantity of FKBP-25 was also associated with the phosphatidylethanolamine-binding protein which is abundant in the brain.     

A novel asparaginase-like protein is a sperm autoantigen in rats

A novel asparaginase-like protein (ALP) of spermatozoa was cloned from rat and human testis cDNA libraries on the basis of reactivity with antibodies produced after vasectomy. Although obstruction of the male reproductive tract is known to cause an immunologic response, few of the sperm antigens responsible for the generation of autoantibodies have been characterized. We are identifying proteins of interest by coring autoantigenic protein spots from two-dimensional (2-D) gels of rat sperm extracts and microsequencing them by mass spectrometry. The peptide sequences from ALP, a 28 kDa, pI 5.7 protein, matched to a single partial length rat EST. These peptide sequences were used to clone a cDNA encoding a novel 333 amino acid open reading frame. The new protein had a similarity to portions of L-asparaginases of plants (43%) and to glycosylasparaginases in animal cells (32%). Human ALP cDNA was subsequently cloned. It showed 77% identity to the rat ALP sequence and the gene, ASRGL1 (asparaginase-like 1), mapped to chromosome locus 11q12.3. Purified recombinant rat ALP (rALP), expressed in E. coli, was used to raise polyclonal antiserum in guinea pigs. Two observations verified that the correct protein had been cloned: 1) the anti-rALP antibody reacted with both rALP and rat sperm; and 2) post-vasectomy sera bound rALP. Anti-rALP antibody stained the midpiece of rat and human sperm coincident with staining by MitoTracker Green FM, suggesting that ALP is associated with the mitochondria. Northern analysis revealed that rat ALP message was abundantly expressed in the testis but was also present in heart, brain, liver, skeletal muscle, and kidney.     

Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ

 Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13–25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35–45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes’ process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins. Accepted: 20 January 1997     

Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells.

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.     

Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen.

The nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic. The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the alpha 3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.     

Antibody reactivities of Mycobacterium paratuberculosis infected sheep as analyzed by enzyme-linked immunosorbent assay and Western blotting

Antibody reactivities in sera from Mycobacterium paratuberculosis (M. ptb) infected and vaccinated sheep were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western (immuno)blotting using a sonicate antigen from M. ptb. Both methods allowed good differentiation between infected/vaccinated animals and noninfected controls. Removal of nonspecific crossreactive antibodies by absorption with a M. phlei sonicate antigen coupled to Sepharose reduced ELISA reactivities of positive sera by 50% and those of noninfected serum by 85%. Immunoblotting analysis revealed that reduction by M. phlei absorption was due to lower reactivities of M. ptb antigens in the range of 30 to 45 kDa. However, one protein with a molecular mass of approx. 27 kDa seemed to be specific for M. ptb since it reacted similarly with nonabsorbed and absorbed serum but not with antibodies which were eluted from M. phlei-Sepharose after absorption. Our findings indicate that M. ptb and M. phlei share a number of common antigens of potential pathogenic importance and that only a smaller part of proteins (i.e. the 27 kDa protein) might be specific for M. ptb.     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

A proteome reference map for Vibrio cholerae El Tor

A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0. The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins. Two strains are compared, strain N16961 and a Latin American El Tor strain C3294. The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium. The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels. The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry. Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins. Two isoforms of OmpU were found. Five operons are proposed and seven hypothetical proteins were experimentally confirmed. Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain. New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.     

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.     

Fast electrotransfer of human serum proteins in their native state from polyacrylamide thin gradient gels reinforced by textiles to polyvinylidene difluoride membranes. Rapid electrotransfer from thin gradient gels reinforced by textiles.

A fast electroblotting technique of native molecules electrophoretically separated in thin (0.25 to 0.5 mm) gradient gels, onto a high capacity membrane of polyvinylidene difluoride is described. Omitting methanol during transfer, the equilibration step is avoided and the same buffer is used in electrophoresis and transfer. As the gel reinforced by fabric never swells nor shrinks, and as all the bands are blotted, the transfer matrix exactly reflects the protein pattern of the original gel. Autoradiography is enhanced and electroelution is homogeneous in all parts of the gels. Significant improvement is noticed in binding proteins of molecular weight from about 20 kDa to more than 700 kDa, as suggested by complete electroelution of all native serum components.     

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.