Kilodalton Protein Research Articles

The human bile salt export pump: Characterization of substrate specificity and identification of inhibitors

Background & Aims: The bile salt export pump (BSEP) is the major bile salt transporter in the liver canalicular membrane. Our aim was to determine the affinity of the human BSEP for bile salts and identify inhibitors. Methods: Human BSEP was expressed in insect cells. Adenosine triphosphatase (ATPase) assays were performed, and bile salt transport studies were undertaken. Results: The BSEP gene, ABCB11, was cloned and a recombinant baculovirus was generated. Infected insect cells expressed a 140-kilodalton protein that was absent in uninfected and in mock-infected cells. An ATPase assay showed BSEP to have a high basal ATPase activity. Transport assays were used to determine the Michaelis constant for taurocholate as 4.25 μmol/L, with a maximum velocity of 200 pmol · min−1 · mg−1 protein. Inhibition constant values for other bile salts were 11 μmol/L for glycocholate, 7 μmol/L for glycochenodeoxycholate, and 28 μmol/L for taurochenodeoxycholate. Cyclosporin A, rifampicin, and glibenclamide were proved to be competitive inhibitors of BSEP taurocholate transport, with inhibition constant values of 9.5 μmol/L, 31 μmol/L, and 27.5 μmol/L, respectively. Progesterone and tamoxifen did not inhibit BSEP. Conclusions: The human BSEP is a high-affinity bile salt transporter. The relative affinities for the major bile salts differ from those seen in rodents and reflect the different bile salt pools. BSEP is competitively inhibited by therapeutic drugs. This is a potentially significant mechanism for drug-induced cholestasis.GASTROENTEROLOGY 2002;123:1649-1658

Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes.

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in Nicotiana benthamiana plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated N. benthamiana leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.

Effects of mutations in the adenoviral E1B 55-kilodalton protein coding sequence on viral late mRNA metabolism.

The human subgroup C adenoviral E1B 55-kDa protein cooperates with the viral E4 Orf6 protein to induce selective export of viral, late mRNAs from the nucleus to the cytoplasm. Previous studies have suggested that such preferential transport of viral mRNA and the concomitant inhibition of export of cellular mRNAs are the result of viral colonization of specialized microenvironments within the nucleus. However, neither the molecular basis of this phenomenon nor the mechanism by which the E1B 55-kDa protein acts has been elucidated. We therefore examined viral late mRNA metabolism in HeLa cells infected with a series of mutant viruses that carry insertions at various positions in the E1B protein coding sequence (P. R. Yew, C. C. Kao, and A. J. Berk, Virology 179:795-805, 1990). All the mutations examined impaired cytoplasmic accumulation of viral L2 mRNAs and reduced L2 mRNA export efficiency. However, in most cases these defects could be ascribed to reduced E1B 55-kDa protein concentration or the unexpected failure of the altered E1B proteins to enter the nucleus efficiently. The latter property, the pleiotropic defects associated with all the mutations that impaired nuclear entry of the E1B protein, and consideration of its primary sequence suggest that these insertions result in misfolding of the protein. Insertion of four amino acids at residue 143 also inhibited viral mRNA export but resulted in increased rather than decreased accumulation of the E1B 55-kDa protein in the nucleus. This mutation specifically impaired the previously described association of the E1B protein with intranuclear structures that correspond to sites of adenoviral DNA replication and transcription (D. Ornelles and T. Shenk, J. Virol. 65:424-439, 1991) and the colocalization of the E1B and E4 Orf6 proteins. As this insertion has been shown to inhibit the interaction of the E1B with the E4 Orf6 protein in infected cell extracts (S. Rubenwolf, H. Schütt, M. Nevels, H. Wolf, and T. Dobner, J. Virol. 71:1115-1123, 1997), these phenotypes provide direct support for the hypothesis that selective viral mRNA export is determined by the functional organization of the infected cell nucleus.

E4orf6 variants with separate abilities to augment adenovirus replication and direct nuclear localization of the E1B 55-kilodalton protein.

The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.

3N0900 349kD接着タンパク質(Gli349)のガラス結合は、マイコプラズマ運動メカニカルサイクルの1ステップである(12.細胞生物学的課題,一般講演,日本生物物理学会第40回年会)

3N0900 349kD接着タンパク質(Gli349)のガラス結合は、マイコプラズマ運動メカニカルサイクルの1ステップである(12.細胞生物学的課題,一般講演,日本生物物理学会第40回年会)

The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity.

The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons. Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5prie; exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles. There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N-terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N. This review will summarize the current information on isoform characteristics of both dUTPase and uracil-DNA glycosylase. It will also focus on evidence for phosphorylation and speculate as to the purpose of these post-translational events.

Molecular and functional analyses of the gene (eshA) encoding the 52-kilodalton protein of Streptomyces coelicolor A3(2) required for antibiotic production.

Analysis of proteins recovered in the S100 precipitate fraction of Streptomyces griseus after ultracentrifugation led to the identification of a 52-kDa protein which is produced during the late growth phase. The gene (eshA) which codes for this protein was cloned from S. griseus, and then its homologue was cloned from Streptomyces coelicolor A3(2). The protein was deduced to be 471 amino acids in length. The protein EshA is characterized by a central region that shows homology to the eukaryotic-type cyclic nucleotide-binding domains. Significant homology was also found to MMPI in Mycobacterium leprae, a major antigenic protein to humans. The eshA gene mapped near the chromosome end and was not essential for viability, as demonstrated by gene disruption experiments, but its disruption resulted in the abolishment of an antibiotic (actinorhodin but not undecylprodigiosin) production. Aerial mycelium was produced as abundantly as by the parent strain. Expression analysis of the EshA protein by Western blotting revealed that EshA is present only in late-growth-phase cells. The eshA gene was transcribed just preceding intracellular accumulation of the EshA protein, as determined by S1 nuclease protection, indicating that EshA expression is regulated at the transcription level. The expression of EshA was unaffected by introduction of the relA mutation, which blocks ppGpp synthesis.

Functions of the adapter protein Cas: signal convergence and the determination of cellular responses.

Since Cas was first identified as a highly phosphorylated 130 kilodalton protein that associated with the v-Src and v-Crk-oncoproteins, considerable effort has been made to determine its function. Its predicted role as a scaffolding molecule based on its domain structure has been largely confirmed. Through its ability to undergo rapid changes in phosphorylation, subcellular localization and association with heterologous proteins, Cas may spatially and temporally regulate the function of its binding partners. Numerous proteins have been identified that bind to Cas in vitro and/or in vivo, but in only a few cases is there an understanding of how Cas may function in these protein complexes. To date, Cas-Crk and Cas-Src complexes have been most frequently implicated in Cas function, particularly in regards to processes involving regulation of the actin cytoskeleton and proliferation. These and other Cas protein complexes contribute to the critical role of Cas in cell adhesion, migration, proliferation and survival of normal cycling cells. However, under conditions in which these processes are deregulated, Cas appears to play a role in oncogenic transformation and perhaps metastasis. Therefore, in its capacity as an adapter protein, Cas serves as a point of convergence for many distinct signaling inputs, ultimately contributing to the generation of specific cellular responses.

A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4 + T lymphocyte responses in B. bovis-immune cattle

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4 + T lymphocytes and IFN-γ production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4 + T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4 + lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4 + T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4 + T cell clones proliferated in response to several B. bovis strains and produced IFN-γ. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/α-crystallin family.

Induction of Cell-Mediated Immunity againstMycobacterium tuberculosisUsing DNA Vaccines Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921-5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2(b)) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8(+) CTL and Th1 responses (high gamma interferon [IFN-gamma], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8(+) CTL and Th1 responses. High levels of IFN-gamma were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.

A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens.

A protein isolated from the supernatant of cryopreserved rooster sperm was found to increase the capability of cryopreserved rooster sperm to bind in vitro to the perivitelline membrane of a chicken egg and substantially raise fertility after artificial insemination (AI). That activity was partially purified and termed universal primary sperm-egg binding protein (UPSEBP). Insufficient protein remained from 6 x 10(11) sperm, despite retention of bioactivity, to allow sequencing. We deduced that the protein may be related to prosaposin (also termed SGP-1, for sulfated glycoprotein-1), and we used published amino acid sequences of prosaposin as a guide for synthesis of peptides. Certain peptides were found to increase in vitro sperm-egg binding and increase fertility of frozen-thawed or fresh rooster sperm, in a manner similar to semipurified UPSEBP. Active epitopes were in a 60 amino acid sequence, reflecting the intervening sequence between saposins A and B, plus short extensions into saposins A and B. Highest activity was found when this synthetic peptide was oxidized to form a disulfide bond between terminal cysteines. Antibody against a synthetic peptide consisting of 58 of these 60 amino acids bound to a 7-9 kilodalton protein in UPSEBP. Collectively, the data support the conclusion that UPSEBP is a fragment of prosaposin. Because prosaposin is in semen in humans and animals, these observations have broad implications for possible cause and therapy of one type of subfertility.

Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein.

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein.

Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells

With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.

Dilated cardiomyopathy is a severe pathology of the heart with poorly understood etiology. Disruption of the gene encoding the negative immunoregulatory receptor PD-1 in BALB/c mice, but not in BALB/c RAG-2-/- mice, caused dilated cardiomyopathy with severely impaired contraction and sudden death by congestive heart failure. Affected hearts showed diffuse deposition of immunoglobulin G (IgG) on the surface of cardiomyocytes. All of the affected PD-1-/- mice exhibited high-titer circulating IgG autoantibodies reactive to a 33-kilodalton protein expressed specifically on the surface of cardiomyocytes. These results indicate that PD-1 may be an important factor contributing to the prevention of autoimmune diseases.

Sucrase-isomaltase is an adenosine 3′,5′-cyclic monophosphate-dependent epithelial chloride channel

We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals. Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody. Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody. Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time.

Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3.

In humans, sterile immunity against malaria can be consistently induced through exposure to the bites of thousands of irradiated infected mosquitoes. The same level of protection has yet to be achieved using subunit vaccines. Recent studies have indicated an essential function for intrahepatic parasites, the stage after the mosquito bite, and thus for antigens expressed during this stage. We report here the identification of liver-stage antigen 3, which is expressed both in the mosquito and liver-stage parasites. This Plasmodium falciparum 200-kilodalton protein is highly conserved, and showed promising antigenic and immunogenic properties. In chimpanzees (Pan troglodytes), the primates most closely related to humans and that share a similar susceptibility to P. falciparum liver-stage infection, immunization with LSA-3 induced protection against successive heterologous challenges with large numbers of P. falciparum sporozoites.

Cloning and characterization of an 18 kiloDalton protein in the roots of the perennial weed Taraxacum officinale Weber (Dandelion) which has allergen- and pathogenesis-related protein properties

The root of the persistent weed, dandelion (Taraxacum officinale Weber), contains a predominant 18 kDa protein which undergoes small seasonal fluctuations in amount, increasing in the late autumn months, and declining in the spring. This protein has been purified and found to consist of two major isoforms of pI 5·56 and pI 5·49. A full-length cDNA has been obtained, coding for the pI 5·56 isoform, and the 156-amino-acid sequence deduced. The protein shows homologies in amino acid composition to several allergen and intracellular pathogenesis-related proteins. The deduced protein does not contain a signal peptide nor any known organelle-targeting sequences, and thus is likely to be cytosolic. Expression of the 18 kDa protein gene is exclusive to the roots and stem tissues; transcripts accumulate during the late autumn months, and decline in the early spring. Changes in the amount of protein in the root are much less. The mRNA for the 18 kDa protein is not present in the dry seed, but appears in the roots within 16–18 h from the start of imbibition, and is expressed constitutively thereafter. Although it is the predominant protein in dandelion roots, its properties are different from those commonly associated with vegetative storage proteins.

“Atypical p-ANCA” in IBD and hepatobiliary disorders react with a 50-kilodalton nuclear envelope protein of neutrophils and myeloid cell lines

Background & Aims: Atypical “antineutrophil cytoplasmic antibodies” (ANCA) are present in patients with ulcerative colitis (UC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). Recently, we showed that atypical p-ANCA react with nuclear envelope proteins of neutrophils. Based on this observation, we aimed to characterize the nuclear antigen recognized by atypical p-ANCA. Methods: We prepared cytoplasmic and nuclear extracts of human neutrophils, human HL-60, and murine 32D myeloid cells. Proteins were resolved by 1- and 2-dimensional gel electrophoresis. Reactive proteins were detected by immunoblotting with sera from 118 individuals (UC, 25; PSC, 28; AIH, 35; disease and normal controls, 30). Atypical p-ANCA (n = 64) were affinity-purified against the reactive protein and investigated for their immunofluorescence pattern using confocal microscopy. Results: Immunoblotting showed reactivity to a myeloid-specific 50-kilodalton nuclear protein with an isoelectric point of pH 6.0 detected in 92% (59 of 64) of the patients with inflammatory bowel or hepatobiliary diseases and atypical p-ANCA. Affinity-purified antibodies against the 50-kilodalton protein gave a nuclear rim-like fluorescence on myeloid cells examined by immunofluorescence microscopy. Affinity-purified antibodies did not recognize antigens in nonmyeloid cells. Conclusions: Atypical p-ANCA in UC, PSC, or AIH recognize a 50-kilodalton myeloid-specific nuclear envelope protein.GASTROENTEROLOGY 2000;119:310-322

A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs.

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at approximately 25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and approximately 20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.