Blot Experiments Research Articles

The occurrence of three isoenzymes of protein kinase C (α, β and γ) in retinas of different species

The localisation and immunochemical identification of 3 different forms of protein kinase C (PKC-α, PKC-β and PKC-γ) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE-Western blotting, respectively. Only in some cases was there a correlation between the findings from each procedure. One reason for the lack of correlation could be the small amounts of PKC present in some retinas, which made detection possible only by first concentrating the antigen by SDS-PAGE and then carrying out Western blotting. Another possible reason is that an antibody recognises unknown antigens immunohistochemically, but, because of their specific characteristics, they are denatured when subjected to SDS-PAGE and Western blotting and therefore remain undetected. PKC-β immunoreactivity is present in rabbit, frog and goldfish retinas but absent from the rat retina. However, SDS-PAGE and Western blotting experiments showed that the PKC-β isoenzyme is absent from the fish retina but present in the rat retina. PKC-β immunoreactivity in rabbit retina is present in ganglion and/or amacrine cells; in the frog retina the enzyme is associated with some bipolar cells. In the goldfish retina, PKC-β is associated with a large population of cells in the ganglion cell layer as well as with some amacrine cell bodies. PKC-α is present primarily in bipolar cells of rat, fish and rabbit retinas and was not detected by immunohistochemistry or blotting experiments in the frog retina. SDS-PAGE and Western blotting of retinal extracts from different species showed that PKC-γ occurs in the rabbit where it was associated with ganglion and/or amacrine cells. While PKC-γ could be detected by blotting experiments in brain extracts from frog, rat and rabbit it was absent from both retina and brain extracts of the goldfish and frog and rat retinal extracts. However, PKC-γ immunoreactivity was associated with many amacrine cells in the frog retina as well as ganglion-like cells in the fish retina.

A nuclear factor that interacts with metal responsive elements of a human metallothionein gene

Metallothioneins (MTs) are low molecular weight heavy metal- binding proteins which are known to play a major role in heavy metal detoxification and understanding of their regulatory mechanism is toxicologically important. Expression of MT genes is induced by heavy metals and metal responsive elements (MREs) upstream of MT genes are essential for the transcriptional activation. By several types of mobility shift assay with 32P-labeled oligonucleotide probes, we detected HeLa cell nuclear as well as cytoplasmic factors that bind to MRE sequences of human MTII A (hMTIIA) gene. One of the nuclear factors, which gives stronger signal than others, was further characterized. Competition experiments showed that the nuclear factor (named MREBP) specifically recognizes MREs of hMTII A gene. EDTA abolished the binding of MREBP to MRE, suggesting that a divalent cation(s) is required for the complex formation. Also in blotting experiments with HeLa nuclear extract and the [ 32P]MRE probes an EDTA-sensitive 95k protein band, which possibly represents MREBP, was detected.

Identification of Giardia lamblia-specific antigens in infected human and gerbil feces by western immunoblotting

Western immunoblot analysis of aqueous extracts of feces obtained from five giardiasis patients and from experimentally infected gerbils (Meriones unguiculatus) with rabbit antiserum to Giardia lamblia cysts has revealed antigens of three molecular weight groups. A stepladderlike, evenly-spaced set of strongly reactive antigens (darkest at a molecular weight [m.w.] of 55,000 to 70,000) appeared in the gerbil feces from day 4 (first experiment) or day 2 (second experiment) and lasted to about day 7 but disappeared completely by day 8 and did not reappear later. These antigenic bands were seen in gerbils infected with two isolates of G. lamblia. These bands were not revealed when antiserum to trophozoites was used as the probe, nor were they evident in specimens from the patients or in a preparation of sonicated cysts. A second group of antigens, represented by two to three low-m.w. bands of approximately 15,000 to 20,000, was evident in both the blots of gerbil feces after approximately day 8 and the specimens from the giardiasis patients. The third group of antigens revealed by blotting experiments was a high-m.w. band (approximately 110,000) which appeared on a number of days (beginning of day 8 of gerbil infection), but this band was not seen in the human specimens. A clear band corresponding to the previously reported GSA-65 antigen was not seen in either the gerbil or the human samples. Some low- and high-m.w. bands were also detected by antitrophozoite serum in the gerbil samples, but these were weak and unimpressive compared with those visualized using anticyst serum. A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay revealed that Giardia spp.-specific stool antigen rose suddenly at day 3 of gerbil infection, at the time when fecal cyst numbers began to rise rapidly.

Solubilization of the 97-kDa native form of liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was partially purified from cholestyramine-fed rats by sequential extraction of the membrane with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and polyethylene glycol nonylphenyl ether (Triton N-101) and solubilized by incorporation of the resulting insoluble protein preparation into a detergent mixture of Triton N-101 and sodium N-lauroylsarcosinate (Sarkosyl) in the presence of high salt. The purification procedure resulted in approximately a 3-4-fold increase in specific activity compared with the microsomal fraction, and the enzyme was recovered with yields as high as 63%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blotting experiment using antiserum to the purified 53,000-dalton reductase fragment showed that the major immunoreactive polypeptide had a Mr of 97,000, that expected for the native intact form of the enzyme (Chin, D. J., Gil, G., Russell, D. W., Liscum, L., Luskey, K. L., Basu, S. K., Okayama, H., Berg, P., Goldstein, J. L., and Brown, M. S. (1984) Nature 308, 613-617). In addition, the effect of various detergents on the activity and stability of the membrane-bound and the partially purified enzyme was determined, and a method for protection of the reductase from inactivation caused by the addition of anionic detergents to the assay mixture is described.

Characterization of pp85, a target of oncogenes and growth factor receptors.

An 85,000-molecular-weight polypeptide (85K polypeptide) has previously been identified as a common substrate for tyrosine phosphorylation upon polyomavirus middle T transformation or upon platelet-derived growth factor stimulation of 3T3 cells. In each case, pp85 has an associated phosphatidylinositol kinase activity. The tissue distribution of pp85 was determined by middle T blotting experiments; the highest levels were found in brain, lung, and spleen tissues. High-resolution examination of 85K by isoelectric focusing demonstrated that there are at least 10 different forms. These were resolved into two families, 85K and 86K; the ratio of the two families changed in different cells. Similar forms were found for pp85 associated with pp60v-src. Individual species within each family differed by phosphorylation. Analysis of pp85 and pp86 by immunoprecipitation with anti-phosphotyrosine antibody showed increasing phosphorylation in response to middle T or pp60v-src transformation. The association of middle T with pp85 and pp60c-src was examined in pulse-chase experiments. Association of middle T with pp60c-src was slow and was accompanied by progressive modification of middle T. pp85 formed a dissociable complex with middle T within 2.5 min.

Characterization of pp85, a Target of Oncogenes and Growth Factor Receptors

An 85,000-molecular-weight polypeptide (85K polypeptide) has previously been identified as a common substrate for tyrosine phosphorylation upon polyomavirus middle T transformation or upon platelet-derived growth factor stimulation of 3T3 cells. In each case, pp85 has an associated phosphatidylinositol kinase activity. The tissue distribution of pp85 was determined by middle T blotting experiments; the highest levels were found in brain, lung, and spleen tissues. High-resolution examination of 85K by isoelectric focusing demonstrated that there are at least 10 different forms. These were resolved into two families, 85K and 86K; the ratio of the two families changed in different cells. Similar forms were found for pp85 associated with pp60v-src. Individual species within each family differed by phosphorylation. Analysis of pp85 and pp86 by immunoprecipitation with anti-phosphotyrosine antibody showed increasing phosphorylation in response to middle T or pp60v-src transformation. The association of middle T with pp85 and pp60c-src was examined in pulse-chase experiments. Association of middle T with pp60c-src was slow and was accompanied by progressive modification of middle T. pp85 formed a dissociable complex with middle T within 2.5 min.

Functional characterization of Tat protein from human immunodeficiency virus. Evidence that Tat links viral RNAs to nuclear matrix.

The processes of transcription and posttranscription are assumed to proceed in close association with the nuclear matrix. In this study we demonstrated that Tat, the trans-activating protein from human immunodeficiency virus type 1 (HIV-1), binds both to the TAR region of the nascent HIV mRNAs and the nuclear matrix with high affinity. Both North/Western blotting experiments and nitrocellulose binding studies revealed that Tat binds with an association constant (K alpha) of approximately 1 x 10(9) M-1 to the TAR segment of HIV RNA; binding of Tat to this sequence which is present between position 32 and 82 downstream from the TATA box was also confirmed by gel retardation assays. Binding of Tat to TAR only occurs if the loop segment in the proposed stem-loop secondary structure of HIV leader mRNA is present. Likewise, Tat binds to the nuclear matrix with a K alpha of 7.5 x 10(7) M-1. The number of binding sites has been estimated to be 2 x 10(8)/micrograms of matrix protein, corresponding to 4 x 10(3) sites/nucleus. Tat displays its bimodal function only in the presence of Zn2+ ions. In vitro transcription experiments, using HIV-1 infected nuclei, demonstrate that beyond the TAR-region HIV RNA synthesis occurs only in the presence of Tat. Present studies indicate that Tat may function as a linker by binding of nascent HIV RNAs to the nuclear matrix.

Monoclonal antibodies used to characterize cDNA clones expressing specific wheat endosperm proteins

A wheat cDNA library, prepared from grain endosperm poly A + -mRNA and cloned into the Escherichia coli expression vector lambda gt11, has been screened with nine monoclonal antibodies having specificities for different wheat endosperm proteins. At least one positive cDNA clone was isolated, and purified, from those selected with each antibody. Each purified cDNA clone was induced to express fusion proteins, and the nitrocellulose membranes to which the proteins were transferred were incubated with each of the other antibodies at two or more concentrations to investigate the extent of homologies between expressed fusion proteins. The specificities of the antibodies were determined using immunoblotting under the same conditions used for binding to the fusion proteins from the expressed cDNA clones. Denatured DNA from each antibody-selected cDNA clone was also characterized by hybridization to αa-/β-genomic gliadin and genomic high molecular weight glutenin subunit DNA probes. Northern hybridizations using the isolated cDNAs as probes for endosperm mRNA were also used to assist clone identification. Some monoclonal antibodies with overlapping specificities (identified from blotting and ELISA experiments) cross-reacted with several expressed cDNA clones. However, in other instances, positive identifications were made of the proteins coded for by single families of the cDNA clones. Monoclonal antibody screening of a wheat cDNA library is useful in identifying families of cDNA clones corresponding to different wheat polypeptides at the primary screening stage, obviating the need in many instances for the application of more tedious methods of clone identification.

Immunological detection of DNA-protein complexes induced by chromate.

A select group of non-histone proteins becomes complexed to DNA after Chinese hamster ovary (CHO) cells are treated with potassium chromate. The most abundant complexed protein has a mol. wt of approximately 45 kd and is thought to be actin. An antiserum to the chromate-induced DNA-protein complexes (DPCs) was prepared to facilitate the study of these complexes. Rather than detecting the predominant silver-stained proteins of DPCs described in an earlier study, this antiserum reacts primarily with an acidic 95-kd protein (p95) that does not silver stain. The antiserum can be used routinely to assay for the induction of p95-DNA complexes produced by chromate and perhaps by other carcinogens. Immunofluorescent staining of CHO cells and immunoblotting of cell fractions show the reactive antigens are within the cell nucleus. Blotting experiments with the antiserum indicate the chromate-induced p95-DNA complex dissociates in the presence of 2-mercaptoethanol, suggesting similarity to the DPCs formed by carcinogenic platinum compounds. A reduced species of chromium (probably Cr3+) may form DPCs by binding to the nitrogen, oxygen or sulfur atoms of proteins and DNA. These results illustrate the usefulness of immunological detection methods to study specific DNA-protein interactions induced by carcinogens. The possible relevance of DPCs in the carcinogenic process is discussed.

Epitope Mapping of the Anti-Urokinase Monoclonal Antibody 5B4 by Isolated Domains of Urokinase

The amino terminal fragment (ATF) of urokinase-type plasminogen activator (uPA) is a degradation product comprising the entire growth factor-like and kringle domains. It has been previously shown that ATF is able to bind to the u-PA receptor through the growth factor-like domain and that the anti u-PA monoclonal antibody 5B4 (Mab 5B4) binds to ATF preventing u-PA receptor binding. To localize more precisely the epitope recognized by Mab 5B4, ATF was subfragmented by controlled enzymatic proteolysis with V8 protease. Three subfragments of 4,000 Mr (F-4k), 11,000 Mr (F-11k) and 12,000 Mr (F-12k) were purified from the reaction mixture and characterized. SDS-PAGE under reducing and non-reducing conditions, N-terminal aminoacid sequence analysis and C-terminal aminoacid analysis of each fragment indicate that F-4k and F-11k correspond to intact growth factor-like domain and kringle domain (residues 4-43 and 44-135 respectively) while F-12k corresponds to the kringle domain cleaved in the first loop at the glu52-gly53 bond. By Western blot and competitive binding experiments we show that Mab 5B4 recognizes an epitope located on the kringle domain of u-PA and that the binding is strongly reduced when the kringle contains an additional cleavage in its first loop. Since the receptor binding site of u-PA has been previously shown to be located on the growth factor-like domain, Mab 5B4 inhibits the binding of uPA to its cellular receptor likely by steric hindrance. Besides the proven utility in epitope localization of anti u-PA monoclonal antibodies, these u-PA fragments may represent powerful tools for studies of structure-function relationship of u-PA.

Expression of intermediate filament proteins during development of Xenopus laevis III. Identification of mRNAs encoding cytokeratins typical of complex epithelia

A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3'-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the 'XK81 subfamily' previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3'-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.

Induction of gamma-interferon by avarol in human peripheral blood lymphocytes.

Avarol is a cytostatic and anti‐human immunodeficiency virus (HIV) agent. In this study, the avarol caused induction of γ‐interferon (IFN‐γ) in buffy coat cells (human peripheral blood lymphocytes) is demonstrated by immunological and molecular biological techniques. IFN‐γ production was detected after a 24‐hr incubation period with avarol; maximal production was obtained after 5 days in the presence of the optimal avarol concentration of 0.75 μg/ml. Blotting experiments using human IFN‐γ cDNA and β‐actin cDNA containing plasmids showed that in the absence of avarol no IFN‐γ transcripts were present in lymphocytes. Already after a 24‐hr incubation with avarol, IFN‐γ gene induction was detected, and maximal induction was found after a 5‐day incubation period. The enhanced IFN‐γ production seems to be caused by a change at the transcriptional and/or post‐transcriptional level, but not during subsequent nucleocyto‐plasmic transport of mRNA. This molecular event is specific, at least in relation to the expression of the β‐actin gene. Our studies demonstrate that avarol displays, besides its potential anti‐tumor and anti‐HIV activity, a potential immunomodulating effect.

Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes.

Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.

Species-specific antigens ofMycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas

Cell proteins from the porcine mycoplasmasMycoplasma hyorhinis, M. hyopneumoniae, andM. flocculare have been analyzed by SDS-gel electrophoresis and immunoblotting. The protein profiles ofM. hyopneumoniae andM. flocculare were similar, but the protein profile ofM. hyorhinis was quite different from the others. Antisera prepared against whole cells of the above three mycoplasmas were used in immunoblotting of electrophoretically separated antigens and in enzyme-linked immunosorbent assay. One major antigen, which had a molecular weight of 73 k, was found to be common to all three mycoplasmas. Another major antigen, with a molecular weight of 41 k, was common toM. hyopneumoniae andM. flocculare and may also be present inM. hyorhinis. Several antigens of comparatively high molecular weights (108 k, 102 k, 93 k, 89 k, and 87 k) seemed to be specific forM. hyopneumoniae. Three antisera prepared by immunization of rabbits with immunoprecipitates obtained by crossed immunoelectrophoresis ofM. hyopneumoniae were also used in blotting experiments. One of these antisera was found to be directed against the 73 k antigen common to the three porcine mycoplasmas investigated. The other two antisera were directed againstM. hyopneumoniae-specific antigens with molecular weights of 74 k, 58 k, 45 k, 44 k, and 38 k.

Double replica electroblotting by oscillating electrotransfer

AbstractProtein blotting has become an indispensable tool in biochemistry and in many blotting experiments it is desirable to obtain more than one replica from the gel, which would permit the use of different probes or staining of one membrane for protein and using the other(s) for probing. This has proved difficult. The different replicas, which can be produced by existing techniques, are far from identical. However, by using oscillating electrotransfer “double replica electroblotting”, two replicas can be obtained from one gel. The two replicas can be made almost identical by reversing the direction of the current in such a way that the efficient transfer time is increased with each period until the gel is almost completely depleted of proteins. The technique is useful for comparison of the reactions of different antisera with one or several antigen samples. Double replica electroblotting has been utilized for ordinary sodium dodecyl sulfate‐polyacrylamide gels, for two‐dimensional polyacrylamide gels and for thin gradient gels analyzed with the PhastSystem (Pharmacia). Double replica electroblotting can, in principle, be used with any blotting system where transfer from the gel to the new medium is achieved by electrophoresis, except in semi‐dry electroblotting with a discontinuous buffer system.

Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies

Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F 1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, ϵ. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 m m Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da ϵ subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, ϵ was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the ϵ was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of ϵ to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which ϵ subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F 1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual α subunit and fragments by an anti-α monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme.

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.

Thirty-Six Human Monoclonal Immunoglobulins With Antibody Activity Against Cytoskeleton Proteins, Thyroglobulin, and Native DNA: Immunologic Studies and Clinical Correlations

AbstractSix hundred and twelve monoclonal Ig (MIg) were studied for their antibody activity against the following autoanti-gens: actin. tubulin, thyroglobulin, myosin, myoglobin, fetuin. albumin, transferrin, and double-stranded DNA (dsDNA). Of these 612 MIg, 36 (i.e., 5.75%) were shown to possess antibody activity. Thirty-two of these 36 (5.22% of the total) were mainly directed against actin. The four others were directed, respectively, against tubulin, myosin, thyroglobulin, and dsDNA. The interaction of the MIg with the respective antigen was demonstrated by immunoenzy-matic methods with monospecific antisera and by blotting experiments. Furthermore, this interaction in the 12 cases studied was mediated by the dimeric fragment F(ab')2 of the MIg. The MIg with antitubulin, antithyroglobulin, and anti-dsDNA activities were exclusively inhibited by their homologous antigens. Those with antiactin activity were predominantly inhibited by actin and also by tubulin and thyroglobulin. The one binding to myosin was, for the most part, inhibited by myosin and also significantly by actin and tubulin. Retrospective clinical analysis was possible for 31 /36 patients. Twenty-six of 31 had malignant lympho-plasmocytic disorders. The five others were followed for miscellaneous disorders without overt signs of multiple myeloma (MM) or Waldenström's macroglobulinemia (WM). The correlation between the antibody activity of the MIg and the clinical features is discussed. These results indicate that a high proportion of MIg possess antibody activity against actin (5.22%). This incidence contrasts sharply with the positive reactions found toward all the other antigens tested: only one each for dsDNA, tubulin, thyroglobulin, and myosin, and none against myoglobin, fetuin, albumin, and transferrin. The significance of these results and the relationship between MIg and natural antibodies are discussed.

Thirty-six human monoclonal immunoglobulins with antibody activity against cytoskeleton proteins, thyroglobulin, and native DNA: immunologic studies and clinical correlations

Six hundred and twelve monoclonal Ig (MIg) were studied for their antibody activity against the following autoantigens: actin, tubulin, thyroglobulin, myosin, myoglobin, fetuin, albumin, transferrin, and double-stranded DNA (dsDNA). Of these 612 MIg, 36 (i.e., 5.75%) were shown to possess antibody activity. Thirty-two of these 36 (5.22% of the total) were mainly directed against actin. The four others were directed, respectively, against tubulin, myosin, thyroglobulin, and dsDNA. The interaction of the MIg with the respective antigen was demonstrated by immunoenzymatic methods with monospecific antisera and by blotting experiments. Furthermore, this interaction in the 12 cases studied was mediated by the dimeric fragment F(ab')2 of the MIg. The MIg with antitubulin, antithyroglobulin, and anti-dsDNA activities were exclusively inhibited by their homologous antigens. Those with antiactin activity were predominantly inhibited by actin and also by tubulin and thyroglobulin. The one binding to myosin was, for the most part, inhibited by myosin and also significantly by actin and tubulin. Retrospective clinical analysis was possible for 31/36 patients. Twenty- six of 31 had malignant lymphoplasmocytic disorders. The five others were followed for miscellaneous disorders without overt signs of multiple myeloma (MM) or Waldenstrom's macroglobulinemia (WM). The correlation between the antibody activity of the MIg and the clinical features is discussed. These results indicate that a high proportion of MIg possess antibody activity against actin (5.22%). This incidence contrasts sharply with the positive reactions found toward all the other antigens tested: only one each for dsDNA, tubulin, thyroglobulin, and myosin, and none against myoglobin, fetuin, albumin, and transferrin. The significance of these results and the relationship between MIg and natural antibodies are discussed.

Establishment of a human keratinocyte cell line carrying complete human papillomavirus type 1 genomes: lack of vegetative viral DNA synthesis upon keratinization.

A human papillomavirus type 1 (HPV-1)/pBR322 recombinant plasmid was constructed consisting of two complete HPV-1 genomes in a head-to-tail arrangement, inserted into the BamHI site of pBR322. To obtain established human keratinocytes keratinocytes carrying dimeric HPV-1, origin-minus simian virus 40 (SV40) DNA was used as a dominant transforming marker in co-transfection experiments performed with cultured human foetal keratinocytes. Using the Southern blotting technique, HPV-1 DNA was detected in one out of five SV40-transformed human keratinocyte cell lines which were obtained in this way. Further blotting experiments using SV40, pBR322 and HPV-1 DNA as probes revealed patterns of hybridization which were consistent with co-integration of SV40 DNA with between two and four copies of the HPV-1 genome. The HPV-1 sequences in this cell line were virtually non-methylated and were transcribed at a lower level than SV40 mRNAs in the same cultured cells. A low level of monomeric HPV form I DNA was detected by blotting of undigested total cellular DNA. No evidence for a stimulation of form I HPV DNA replication could be obtained by blotting analysis of total DNA extracted from keratinizing cysts, which were formed by subcutaneous inoculation of these cells into nude mice.