Detection of multiple antigenically related proteins from various rat tissues by monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase.

Mouse hybridomas were prepared by fusing myelomas and spleen cells from mice immunized with purified rat 3 alpha-hydroxysteroid dehydrogenase. Hybridomas secreting monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase were selected by indirect enzyme-linked immunoassay and then subcloned by limiting dilution. From two mice we have obtained four positive hybridomas, three secreting high affinity immunoglobulin (Ig) G1 and one secreting IgM. Only two of these monoclonal antibodies (MAbs 3G6 and 7D3, both IgG1) recognized denatured enzyme and, therefore, were used for further immunoblotting experiments. MAb 7D3 recognized a structurally related mouse enzyme, but not the human enzyme, whereas monoclonal antibody 3G6 recognized a human enzyme, but not the mouse enzyme. When these two monoclonal antibodies were used in immunoblotting to survey the expression of 3 alpha-hydroxysteroid dehydrogenase in rat liver and a number of other tissues, striking differences were found in the protein band patterns in kidney, lung, and testis. Both MAbs 7D3 and 3G6 recognized 3 alpha-hydroxysteroid dehydrogenase, a 34-kDa 7D3 recognized a protein of the same size as the liver protein, whereas MAb 3G6 recognized a 34-kDa protein plus another protein of 36 kDa. In kidney only MAb 3G6, but not MAb 7D3, recognized a 34-kDa protein. Conversely, the 34-kDa protein in testis was recognized by MAb 7D3, but not by MAb 3G6. These findings suggest the existence of multiple antigenically related proteins in different tissues.     

Immunological Evidence of Connexin-like Proteins in the Plasma Membrane of Vicia faba L.

Plasma membranes from three week old leaves of Vicia faba L. were enriched by aqueous two-phase partitioning to high purity. Plasma membrane proteins were immunoblotted with polyclonal, monospecific antibodies raised against mouse liver connexins (cx) 32 and 26. Immunostaining after treatments with cx 32 antibodies revealed the existence of a 29 kDa protein, clearly enriched in the plasma membrane fraction. An additional immunoreactive band of 20 kDa, possibly a degradation product of the 29 kDa protein, was found in the soluble fraction. When immunoblots were incubated with cx 26 antibodies, a 40 kDa band with a strong immunoresponse appeared, assumed to present the dimeric form of a 21 kDa, cx 26-like plant protein. The monomeric form could be only obtained when intact leaf material or mesophyll protoplasts from three week old plants were directly SDS-extracted. Furthermore, in young, one week old leaves, the monomer seems to exist in larger amounts, together with another crossreacting 35 kDa protein. The 29 kDa (cx 32-related) as well as the 40 kDa (cx 26-related) polypeptide is obviously located in the plasma membrane. The 40 kDa protein has to be considered as a new connexin-like plant protein.     

Swim bladder gas gland cells produce surfactant: in vivo and in culture

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.     

The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally.

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.     

Production and Characterization of Monoclonal Antibodies to Bovine Brain Succinic Semialdehyde Reductase

Abstract: Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced and characterized. A total of nine monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which two inhibited the enzyme activity and three stained cytosol of rat spinal cord neurons as observed by indirect immunofluorescence microscopy. When unfractionated total proteins of bovine brain homogenate were separated by gel electrophoresis and immunoblotted, the antibodies specifically recognized a single protein band of 34 kDa, which comigrates with purified bovine succinic semialdehyde reductase. Using the antisuccinic semialdehyde reductase antibodies as probes, we investigated the cross-reactivities of brain succinic semialdehyde reductases from some mammalian and an avian species. The immunoreactive bands on western blots appeared to be the same in molecular mass—34 kDa—in all animal species tested, including humans. The result indicates that brain succinic semialdehyde reductase is distinct from other aldehyde reductases and that mammalian brains contain only one succinic semialdehyde reductase. Moreover, the enzymes among the species are immunologically very similar, although some properties of the enzymes reported previously were different from one another.     

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component of the intracellular inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we show that both monoclonal (60019-2-Ig) and polyclonal (10782-2-AP) anti-TDP-43 antibodies recognize amino acids 203-209 of human TDP-43. The monoclonal antibody labeled human TDP-43 by recognizing Glu204, Asp205 and Arg208, but failed to react with mouse TDP-43. The antibodies stained the abnormally phosphorylated C-terminal fragments of 24-26 kDa in addition to normal TDP-43 in ALS and FTLD brains. Immunoblot analysis after protease treatment demonstrated that the epitope of the antibodies (residues 203-209) constitutes part of the protease-resistant domain of TDP-43 aggregates which determine a common characteristic of the pathological TDP-43 in both ALS and FTLD-TDP. The antibodies and methods used in this study will be useful for the characterization of abnormal TDP-43 in human materials, as well as in vitro and animal models for TDP-43 proteinopathies.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassay

Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung.     

Purification of T47D human progesterone receptor and immunochemical characterization with monoclonal antibodies

In order to obtain steroid-independent probes for human progesterone receptor (PR), the A [88-93 kilodalton (kDa)] and B (109-119 kDa) forms of PR from T47D human breast cancer cells were partially purified and used to generate a series of 14 monoclonal antibodies. Initially, unoccupied PR was isolated from cytosol extracts by steroid affinity chromatography, followed by chromatography on diethylaminoethyl Bio-Gel. The partially pure (3-15%) PR consisted of two steroid-binding components that migrated at 89 kDa and 109 kDa in reducing sodium dodecyl sulfate gels after being photoaffinity labeled with the synthetic progestin [3H]R5020. Two unique monoclonal antibodies to PR were derived from a male Lewis rat immunized with this material. One of these antibodies (JU601) was coupled to Sepharose 4B and used to purify T47D nuclear PR for additional immunizations. Highly purified (30-70%) PR migrated as 93 kDa and 119 kDa progestin-binding proteins in sodium dodecyl sulfate gels. In all, thirteen monoclonal antibodies were obtained that recognized epitopes shared by both receptor forms. One mouse immunoglobulin G (KC146) was completely specific for the larger B form. Interestingly, the epitope for this antibody was present on all PRs tested, including the B form of PR from chicken oviduct, whereas nine other antibodies recognized only human PR and the remaining four cross reacted with rabbit PR. With the exception of the JU145 and JU601 rat immunoglobulin Ms, all antibodies appeared to be completely specific for the A or B forms of PR. Each recognized the cytosol and nuclear forms of occupied as well as unoccupied PR. Although the relationship between B and A was not established, it is clear that an amino-terminal region of B is not present in A, and that a significant portion of A and B are either identical or very similar in amino acid sequence.     

Purification and molecular identification of the human hyaluronan receptor for endocytosis

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.     

Mechanism of interferon action. Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1 were prepared using protein P1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P1 recognized with comparable efficiency P1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P1 and the alpha subunit of initiation factor eIF-2. Three BALB/C mouse monoclonal antibodies efficiently recognized human protein P1, but either did not recognize or recognized very poorly P1 from mouse cells. A fourth monoclonal antibody against human P1 recognized mouse P1 with nearly equal efficiency. Immunoprecipitation of human P1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P1 may exist.     

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.     

Identification of human, mouse, and rat retinoic acid receptor alpha using monoclonal antibodies.

Monoclonal antibodies that recognize the human, mouse, and rat retinoic acid receptor alpha (RAR alpha) protein have been generated using synthetic peptides. Less well-characterized monoclonal antibodies were also generated against the RAR beta and RAR gamma proteins. Monoclonal antibodies of the IgG1 (R alpha 10) and IgG2a (R alpha 13) isotypes effectively and specifically recognize both the human and mouse RAR alpha protein. Preincubation of the antibodies with the synthetic RAR alpha peptide, but not with the RAR beta or RAR gamma peptides, blocked recognition of the approximately 55 kDa RAR alpha protein on western blots. These monoclonal antibodies also detected differing levels of RAR alpha in various rat tissues. These monoclonal antibodies will serve as powerful reagents to study the structure and regulation of the retinoic acid receptor protein.     

The phosphorylation of human link proteins

Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.     

Identification of centromere proteins in different mammalian cells

The characterization of centromeric proteins is facilitated using anti-centromere antibodies present in the sera of patients with the CREST variant of scleroderma. We have employed these sera to determine whether or not those proteins are present in different mammalian species, as well as to study their tissue distribution. Here, we describe the immunofluorescent pattern and the proteins recognized by CREST sera in dividing and resting cells from mouse, rat, swine, hamster, rabbit, and man. In nuclear preparations from cultured cells, thymocytes and spermatozoa from these species, the antigens recognized by CREST sera are proteins of 18 to 20 kDa in all species tested, except in rat. Additionally, two peptides of 80 and 140 kDa were observed in human preparations. In contrast, a 50 kDa peptide is the primary protein detected by the sera in rat nuclei.     

Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification, Expression, and Nuclear Location of Murine Mage-b2 Protein, a Tumor-associated Antigen

MAGE-1, which was originally identified by reacting with cytolytic T lymphocytes derived from the blood of melanoma patients, is a member of a gene family consisting of 17 structurally related genes. The MAGE genes are expressed only in the testis among normal tissues and in a number of human tumors of various histological types. Murine MAGE (also called SMAGE or Mage) genes were found in a study aimed at detecting mouse genes homologous to human MAGE genes. However, the biological functions of MAGE and Mage are currently unknown. To understand the biological functions of Mage, in the present study a recombinant SMAGE2 (Mage-b2) protein of 43 kDa was produced and monoclonal antibodies reactive with Mage-b2 protein were generated. One monoclonal antibody, smpG4A, specifically recognized a 43 kDa protein in lysates of Mage-b2 mRNA-positive sarcoma cells and of the testis. Immunohistochemistry showed that Mage-b2 is located in the nucleus of Mage-b2 mRNA-positive sarcoma cells. These results should contribute to understanding the biological functions of Mage.     

Identification, Expression, and Nuclear Location of Murine Mage-b2 Protein, a Tumor-associated Antigen

MAGE-1, which was originally identified by reacting with cytolytic T lymphocytes derived from the blood of melanoma patients, is a member of a gene family consisting of 17 structurally related genes. The MAGE genes are expressed only in the testis among normal tissues and in a number of human tumors of various histological types. Murine MAGE (also called SMAGE or Mage) genes were found in a study aimed at detecting mouse genes homologous to human MAGE genes. However, the biological functions of MAGE and Mage are currently unknown. To understand the biological functions of Mage, in the present study a recombinant SMAGE2 (Mage-b2) protein of 43 kDa was produced and monoclonal antibodies reactive with Mage-b2 protein were generated. One monoclonal antibody, smpG4A, specifically recognized a 43 kDa protein in lysates of Mage-b2 mRNA-positive sarcoma cells and of the testis. Immunohistochemistry showed that Mage-b2 is located in the nucleus of Mage-b2 mRNA-positive sarcoma cells. These results should contribute to understanding the biological functions of Mage.