Monoclonal antibody studies of alpha-keratin low-sulfur proteins

Monoclonal antibodies to the two families of low-sulfur proteins from wool were produced. Selection was applied to identify those hybridomas secreting antibody that was effective in the Electro-blot system. The specificities of eight different monoclonal antibodies were investigated by their binding to alpha-keratin low-sulfur proteins which had been subjected to electrophoresis from wool, goat hair, porcupine quill, rat hair and echidna quill, using the Electro-blot procedure. Considerable cross-reactivity was found both within the low-sulfur protein components of individual keratins from a particular species, and also between the keratins of the different species. Some antibodies were found to bind selectively to components of one family of low-sulfur proteins in wool, while others recognized determinants in both families, indicating some homology between the two families.     

Structural homology between hard α-keratin and the intermediate filament proteins desmin and vimentin

Although it has been assumed that the microfibrils in hard -keratin are members of the class of structures known as intermediate filaments (IF), no firm chemical evidence relating the low-sulfur proteins in hard -keratin to other IF proteins has yet been published. We now present primary sequence data for two components from wool keratin which show striking similarities with two IF proteins, desmin and vimentin. The sequences show marked homology, a heptad repeat and a 9.5-residue periodicity in the linear disposition of the acidic and the basic residues. These data thus provide the first evidence that the low-sulfur proteins in hard -keratin and the other IF proteins do indeed have both a similar structure and a common evolutionary origin.     

Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the ascidian Halocynthia roretzi

To obtain specific immunological probes for studying molecular mechanisms involved in the early embryonic development of ascidians, we have produced monoclonal antibodies directed against a homogenate of larvae of the ascidian Halocynthia roretzi. Among these, we have screened monoclonal antibodies that specifically recognize cells and/or tissues of the embryo. Characterization of six epidermis-specific monoclonal antibodies (including larval tunic-specific and larval fin-specific), three muscle-specific antibodies, two endoderm-specific antibodies, one notochord-specific antibody and two monoclonal antibodies that specifically recognize trunk-lateral cells suggests that these monoclonal antibodies may be useful as markers for analysing molecular mechanisms involved in specification of these cells. Seven monoclonal antibodies characteristically stain intercellular materials of the developing embryo and may therefore be valid for studying cellular construction of the embryo. Furthermore, monoclonal antibodies that recognize components of follicle cells, perivitelline space and sperm have also been established.     

Monoclonal antibodies to a subfraction of merino wool high-tyrosine proteins

Monoclonal antibodies were prepared which react with members of the high-tyrosine type proteins from Merino wool. Specificity was confirmed by the use of Western transfer immunoassays and by enzyme-linked immunosorbent assay on purified fractions. Immunofluorescent staining of sections of wool follicles using the antibodies showed that the proteins were present in the developing wool shaft but that staining was asymmetric, indicating specific location of the proteins in the orthocortex of the fibres. Immunogold-electron microscopy confirmed that one of the antibodies bound to the keratin microfibril bundles.     

Characterization of a monoclonal antibody (SBU-1) made to the thymic rudiment of sheep

A monoclonal antibody (SBU-1) was raised to sheep thymic rudiment by fusion of NSI myeloma cells with spleen cells from BALB/c mice immunized with thymic rudiment isolated from fetal sheep between 25-30 days of gestation. By employing the indirect immunoperoxidase technique the antigen recognized by SBU-1 was found to be present in the epithelial reticular cells of the fetal sheep thymus. The intensity of staining decreased as gestation progressed. In the adult thymus the antigen was mainly restricted to Hassall's corpuscles and occasional epithelial cells in the medulla. In addition, the antigen was also shown to be present in epithelial cells of the small intestine, the bronchiole, the keratinized epithelium of the rumen, and the epithelial cells of the kidney tubules. By use of immunofluorescence the antigen was shown to be present in most of the cells of wool follicles and the cortex of developing wool fibers. Western blotting of SBU-1 against the low-sulfur alpha-keratin proteins of wool confirmed that the antigen recognized by SBU-1 belongs to a family of keratins. It was concluded that SBU-1 was raised against alpha-keratin expressed by the epithelial cells of the thymic rudiment and that the expression of this antigen on the reticular network of the thymus declined with advancement of pregnancy.     

Immunochemical characterization of related families of glycoproteins in desmosomes

Using several biochemical approaches, we have characterized the relatedness of the various glycoprotein components of the bovine epidermal desomosome. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of purified epidermal desmosomes reveals 12 proteins, of which 8 are glycosylated. Analysis with monoclonal antibodies indicates that the 8 glycoproteins comprise 3 antigenically distinct protein families. Members of the highest molecular weight glycoprotein family (a triplet of Mr = 150,000) were not distinguishable by partial proteolytic peptide mapping. At least 6 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. Members of a 97,000-118,000-dalton glycoprotein family (about 4 bands) generate very similar but not identical partial proteolytic peptide maps. At least 3 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. A Mr = 22,000 glycoprotein is immunologically unrelated to either of the high molecular weight glycoprotein families. Lectin-binding profiles indicate that within each immunologically related family the glycoproteins are similar in their oligosaccharide composition. Some lectins distinguish among the families. These glycoproteins probably mediate the specific intercellular recognition and adhesive functions of the desmosome.     

Characterization of cell surface glycoproteins recognized by the granulocyte-specific monoclonal antibody, AHN-1

Major surface-iodinated proteins of Mr 105,000 and 145,000 of normal human neutrophils are immunoprecipitated by a number of monoclonal antibodies (AHN-1 to AHN-6), which react specifically with granulocytes among peripheral blood cells and selectively inhibit phagocytosis. These proteins, and an Mr 60,000 component, were purified by monoclonal antibody affinity chromatography, molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Each of the three purified proteins was immunoprecipitated by all six antibodies. Nevertheless, tryptic peptide maps of the three proteins indicated that each was a distinct component. AHN-1 to AHN-6 also bound to glycolipid fractions of human neutrophils, and the binding of each antibody to human neutrophils was blocked by the carbohydrate sequences, lacto-N-fucopentaose III. The data indicate that a predominant antigenic determinant of human neutrophils is lacto-N-fucopentaose III, or related carbohydrates, present on three distinct proteins as well as glycolipids. At least one of these molecules appears to be involved in the process of phagocytosis.     

Fine oral filaments in Paramecium: a biochemical and immunological analysis

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.     

Human IgM monoclonal proteins that bind 3-fucosyllactosamine, asialo-GM1, and GM1

Analysis of monoclonal human Ig that occur in association with lymphoproliferative diseases has provided valuable information about antibody structure and idiotypes. We analyzed 940 human sera that contained monoclonal IgM proteins for their ability to bind to four carbohydrate epitopes. Ten sera bound asialo-GM1, five of these sera also bound GM1, 10 bound to 3-fucosyllactosamine (3-FL), and one each bound to levan and galactan. Although the antibody activity in each serum was associated with a single L chain isotype, both kappa and lambda isotypes were represented among the proteins that bound to asialo-GM1 and to 3-FL. Some antibodies against asialo-GM1 were highly specific for this compound, whereas others cross-reacted with the structurally related gangliosides GM1 and GD1b. The antibodies to asialo-GM1 also varied considerably in their ability to lyse liposomes that contain asialo GM1. An association of IgM mAb against gangliosides with peripheral neuropathies has been reported recently, but only one of five patients whose antibodies reacted with GM1 ganglioside had a neuropathy. The antibodies that bound 3-FL exhibited narrower specificity, and less than 10% cross reactivity was noted with structurally related carbohydrates. The frequency of monoclonal proteins that bound 3-FL and asialo-GM1, approximately 1:100 sera for each specificity, was surprisingly high in view of the fact that both of these epitopes are expressed in human tissues. We suggest that these antibodies may be poly-specific and/or that the subset of B lymphocytes that synthesizes these anti-carbohydrate antibodies undergoes malignant transformation more frequently than other B lymphocytes.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.     

Identification of microRNAs in Wool Follicles during Anagen,Catagen, and Telogen Phases in Tibetan Sheep

Background

Wool quality is one of the most important economic traits in sheep. The wool fiber is derived from specialized skin cells that are referred to as wool follicles. To understand the roles of microRNAs (miRNAs) in wool fiber growth, we detected the expression patterns of miRNAs in wool follicles at the anagen, catagen, and telogen stages from Tibetan sheep through Solexa sequencing.

Results

A total of 244 mature miRNAs were identified. Of these, only five miRNAs are listed in the database of sheep miRNAs (miRBase Database V19), and the other 239 miRNAs have not been previously described in this species. Further analyses indicated that 204 miRNAs are evolutionarily conserved among mammal species, whereas 35 of the identified miRNAs were first found specifically in sheep. The expression pattern analyses showed that the expression levels of 39, 34, and 20 of the miRNAs significantly change between anagen and catagen, between anagen and telogen, and between catagen and telogen, respectively. The results of the bioinformatics analysis show that these differentially expressed miRNAs might regulate wool follicle development by targeting genes in many different pathways, such as the MAPK and Wnt pathways, as well as the pathways that regulate the actin cytoskeleton, focal adhesion, and tight junctions. Furthermore, we identified six differentially expressed miRNAs (oar-miR-103-3P, oar-miR-148b-3P, oar-miR-320-3P, oar-miR-31-5P, oar-novel-1-5P, and oar-novel-2-3P) that might target the key genes of the Wnt pathway. It has been reported that the Wnt pathway is critical for wool follicle development. Therefore, these miRNAs may regulate wool development through the Wnt pathway.

Conclusions

Our results provide new information on the identification and expression pattern of miRNAs in wool follicles. Our data might therefore aid in the understanding of the mechanisms of wool follicle development in sheep.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Developmental stage-specific antigens in the nervous system of the cockroach

A specific effort was made to obtain monoclonal antibodies that bind to macromolecules that play a role in the development of the nervous system. It was considered that good candidates for such molecules were those that were only transiently present in the embryonic nervous system. Hybridomas were prepared from spleen cells taken from mice that had been immunized with nerve cords from cockroach embryos at the 43-50% stage of development. The hybridoma supernatants were screened for antibody binding to frozen sections of both embryonic and adult thoracic ganglia. Cell lines that produced monoclonal antibodies that transiently bound to the embryonic nervous system were saved and cloned. These developmental stage-specific monoclonal antibodies either did not bind to the adult nervous system or bound to it with a pattern very different from that in the embryonic nervous system. The developmental stage-specific antigens detected by these monoclonal antibodies were organized into four categories based on the part of the embryonic nervous system in which they were transiently localized. These include binding to the cell bodies of all neurons, cell bodies of subsets of neurons or neuroblasts, subsets of axons, and the neuropile. Preliminary biochemical characterization of the antigens showed that many of these antibodies were recognizing carbohydrate epitopes. Functions for these antigens, most of which are components of the cell surface, are tentatively proposed.     

New proteins related to the Ser-Arg family of splicing factors.

A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. Using a selective precipitation method, more than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Iariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

The pharmacological characteristics, immunochemical identification and study of the location of quisqualate receptors in the rat and human brains]

The kinetics of [3H]-L-glutamate binding to brain synaptic membranes (SM) and to glutamate-binding proteins (GBP) was determined with agonist and monoclonal antibodies (MAbs). It was revealed, that rat and human brain GBP have individual protein components with M(r) from 14 to 92 kDa. Quisqualate inhibited [3H]-L-glutamate binding to solubilized and to purified 68 kDa protein component. MAbs have the most activity, and NMDA was failure. It has been shown that 68 kDa component antigen determinants are similar to those of bovine, frog and rat brain synaptic membranes. Anti-GBP monoclonal antibodies blocked functional non-NMDA receptors in isolated frog spinal cord. Immunocytochemistry was done on rat and human brain sections. Distribution of quisqualate receptors was determined with light and electron microscopy. Some properties of vertebrate CNS non-NMDA receptors are discussed.     

Bony Fish Myelin: Evidence for Common Major Structural Glycoproteins in Central and Peripheral Myelin of Trout

Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two-dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IP0. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-actinin is a component of the Z-filament, a structural backbone of skeletal muscle Z-disks

The relationship between the ultrastructure and protein components of Z-disks was studied using psoas muscles of rabbit and breast muscles of chicken. Glycerinated fiber bundles of these muscles were treated with a solution containing 0.1 mM Ca2+ to induce a structural weakening of Z-disks non-enzymatically. During the treatment, clear geometrical configurations of Z-filaments could be observed along with removal of amorphous matrix under an electron microscope. Even after a prolonged treatment for 14 d in which all Z-disks were entirely weakened, entangled Z-filaments were left in the original region of the disks. Immunoelectron microscopic observations showed that antibodies against alpha-actinin bound to the entangled Z-filaments, forming dense lines at the position of the original Z-disks. On SDS-PAGE of Z-disk substances, alpha-actinin remained unchanged and Mr 75,000 and 55,000 proteins were removed during the Ca2+-treatment. We therefore conclude that alpha-actinin is a component of Z-filaments, and that the amorphous matrix is composed at least of these Mr 75,000 and 55,000 proteins.     

Apolipoprotein D and cross-reacting human plasma apolipoproteins identified using monoclonal antibodies

We have produced five hybridomas which secreted monoclonal antibodies that reacted with human plasma apolipoprotein D. On analysis by polyacrylamide gel electrophoresis (PAGE) high density lipoproteins and lecithin:cholesterol acyltransferase (EC 2.3.1.43)-enriched fractions of plasma contained many protein bands that reacted with the antibodies. Purified apolipoprotein D had the lowest Mr (29,000), the lowest pI (4.8-5.2), and the greatest migration on alkaline urea-PAGE of all the immunoreactive bands. These characteristics agreed with those described for apolipoprotein D in the literature. The other immunoreactive proteins had apparent Mr from about 39,000 to 98,000, they migrated more slowly than apolipoprotein D on alkaline urea-PAGE, and there were 10 polymorphs on isoelectric focusing. These cross-reacting proteins were present in the high density lipoproteins of each of four individuals sampled on several occasions and in pooled plasma. All of the monoclonal antibodies reacted both with apo-D and the higher Mr cross-reacting proteins. Each of our five monoclonal antibodies bound to one of two distinct antigenic sites on apo-D, determined by antibody competition immunoassays. Neither of these two sites was composed of carbohydrate, but expression of both sites seemed to be influenced by thiol-reducing agents: site 5G10 gained but 4E11 either lost immunoreactivity or was unchanged by reduction according to the conditions. We conclude that apolipoprotein D is only one of several plasma proteins, which contain two homologous polypeptide antigenic sites, recognized by monoclonal antibodies and also by a specific goat antiserum. Apolipoprotein D had the least Mr of these proteins.