Change in phenylalanine ammonia lyase activity and isozyme patterns of polyphenol oxidase and peroxidase by salicylic acid leading to enhance resistance in cowpea against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

In this paper, we describe a study concerning the determination of phenylalanine ammonia lyase (PAL) activities leading to decline in disease progress caused by Rhizoctonia solani after application of salicylic acid (SA). Two applications of 1.4 mM SA (pH 6.5) followed by inoculation with Rhizoctonia solani resulted in a quantitative change in polyphenol oxidase (PPO), peroxidase (PRX) isoforms and increase in PAL activities from 4.38 to 19.48 unit g⁻¹ (FM) h⁻¹ in Bundel-1, UPC-4200 and IFC-902 cowpea genotypes. Increase in PAL activities was further observed specifically in UPC-4200 when plants were exposed with Rhizoctonia solani spores. Total soluble proteins did not change after SA treatment, however they were significantly increased in SA sprayed-inoculated UPC-4200 genotype. Of the ten detected isoforms of polyphenol oxidase, isoforms 7 and 10, and isoform 4 of peroxidase showed increased activities by SA application. The disease symptoms measured as areas under progress curve (AUDPC) indicated less A value in SA sprayed Bundel-1 and UPC-4200 genotypes over their controls.     

Production of antisera specific for idiotype(s) of murine anti-(T,G)-A--L antibodies.

Antibodies to the synthetic polypeptide (T,G)-A--L were raised in C57BL/10 and C3H.SW mice. For each strain, the anti-(T,G)-A--L antibodies from 10 animals were pooled, affinity purified on a (T,G)-A--L-Sepharose column, and used to immunize Lewis rats. The resulting rat antisera were adsorbed with insolubilized normal mouse globulin in order to remove anti-isotypic and anti-allotypic antibodies. The residual antibodies specifically inhibited the binding of (T,G)-A--L by anti-(T,G)-A--L as measured by a radioimmunoassay. The specificity of this inhibition was demonstrated as follows: 1) failure of the anti-(T,G)-A--L anti-idiotype to inhibit the binding of nuclease to anti-nuclease antibody of the same allotype; 2) failure of Lewis anti-[B10 anti-(T,G)-A--L] to inhibit C3H.SW anti-(T,G)-A--L and vice versa; 3) ability to absorb anti-C3H.SW anti-idiotypic activity on insolubilized C3H.SW anti-(T,G)-A--L but not on B10 anti-(T,G)-A--L. The same or cross-reactive idiotype(s) was present in the majority of individuals of each of these strains.     

Immunochemical analysis of subunit structures of 1,4-dihydropyridine receptors associated with voltage-dependent Ca2+ channels in skeletal, cardiac, and smooth muscles

Previous purification studies of the 1,4-dihydropyridine receptor associated with the calcium channel of rabbit skeletal muscle had shown that it is composed of a large glycoprotein of Mr 140,000-145,000 associated with a smaller component of Mr 32,000-34,000. Specific antisera have now been prepared against the larger component (anti-140 serum) and the smaller one (anti-32 serum). The specificity of these two antisera has been analyzed by immunoblot assays with microsomal preparations of rabbit skeletal muscle. Under disulfide-reducing conditions the anti-140 serum specifically labeled a polypeptide of Mr 140,000 while the anti-32 serum labeled three polypeptides of Mr 32,000, 29,000, and 26,000. Under nonreducing conditions both the anti-140 and the anti-32 sera specifically recognized a single large polypeptide of Mr 170,000. The same type of approach showed that the dihydropyridine receptor in cardiac and smooth muscles had a polypeptide composition similar to that found in skeletal muscle with a large polypeptide of Mr 170,000-176,000 made of two different chains of about Mr 140,000 and 34,000-32,000 associated by disulfide bridges.     

LOCALIZATION OF THE THY-1 ANTIGEN IN RAT BRAIN AND SPINAL CORD BY IMMUNOFLUORESCENCE

Abstract— The Thy-1 antigen of rat brain is a membrane glycoprotein of molecular weight 17,500. It was localized in sections of brain and spinal cord by indirect immunofluorescence using rabbit antisera raised against purified Thy-1 and fluorescein conjugated purified sheep F(ab')₂, anti-(rabbit IgG) antibody fragments. The specificity of the anti-(Thy-1) sera was tested by a quantitative indirect radioactive binding assay which is particularly useful for ascertaining the specificity of reagents used in immunohistochemical studies. Purified Thy-1 was used to absorb the anti-(Thy-1) sera for controls in the immunofluorescence experiments. Strong specific fluorescence was found throughout the gray matter of brain and spinal cord with lesser amounts in white matter. The nuclei of all neural cells and also myelin lacked fluorescence. Some of the large neurons contained weak cytoplasmic fluorescence, but the majority of the immunofluorescence was located in the neuropil of the brain and spinal cord. There was an indication that Thy-1 was associated with synaptic knobs due to its presence in synaptic glomeruli and its granular appearance around some neurons. An additional association with glial membranes could not be excluded.     

Idiotypic analysis of hybridoma antibodies to branched synthetic polymer (Tyr, Glu)-Ala-Lys: idiotypic relationship with antibodies to linear random polymer (Glu60, Ala30, Tyr10)

The expression of three anti-GAT idiotypes, CGAT, Gte, and GA-1, on 17 C57BL/10 and four C3H.SW hybridoma anti-(T,G)-A--L antibodies was analyzed. These hybridoma anti-(T,G)-A--L antibodies exhibited two patterns of fine antigen binding specificity. The majority of the hybridoma antibodies bound the (T,G)-A--L, GT, and GAT polymers but not the GA polymer, and were designated as GT-reactive hybridoma antibodies. A minor population of hybridoma anti(T,G)-A--L antibodies bound to (T,G)-A--L but not to GT, GAT, or GA, i.e., (T,G)-A--L-specific. A complete correlation between fine antigen binding pattern and the expression of CGAT idiotype was demonstrated. None of the 21 hybridoma anti-(T,G)-A--L antibodies expressed the GA-1 idiotype. All of the GT-reactive and none of the GT-nonreactive hybridoma anti-(%,G)-A--L antibodies expressed the CGAT idiotype. Furthermore, the Gte idiotype was found on the majority of CGAT+-bearing C57BL/10 hybridoma anti-(T,G)-A--L antibodies. These results indicate that C57BL/10 anti-(T,G)-A--L antibody repertoire can be grouped into a minimum of three families; i.e., CGAT+ Gte+, CGAT+ Gte-, and CGAT- Gte- families, with the CGAT+ Gte+ family as the major compartment. This is confirmed by the high percentage idiotype binding of serum anti-(T,G)-A--L antibodies with anti-CGAT idiotypic antisera. Finally, anti-idiotypic antisera made against CGAT+ hybridoma anti-GAT or anti-(T,G)-A--L antibodies crossreact extensively with other CGAT+ hybridoma anti-GAT and anti-(T,G)-A--L antibodies. However, additional experiments demonstrated that CGAT+ hybridoma anti-(T,G)-A--L antibodies also possess private idiotypes.     

Macrophage-lymphocyte interaction. III. Site of alloantiserum inhibition of T lymphocyte proliferation induced by allogeneic or aldehyde-bearing cells.

Inhibition by anti-Ia sera of guinea pig T lymphocyte proliferation induced by allogeneic macrophages (MLR) and NaIO4 or neuraminidase-galactose oxidase-treated macrophages has been investigated in order to identify the target cell upon which the antisera act. Anti-2 and anti-13 alloantisera were found to inhibit both MLR and aldehydeinduced T cell reactivity when directed against the specificity of the stimulatory macrophage. Little or no inhibition was observed when these antisera were directed against the T lymphocyte specificity when cultures were harvested at the time of peak proliferation. In addition, anti-2 serum was found to inhibit macrophage-lymphocyte rosett formation at 20 hr between neuraminidase-galactose oxidase-treated strain 2 macrophages and strain 13 lymphocytes. These findings demonstrate that inhibition of T cell proliferation can be produced by anti-Ia sera directed against the macrophage and raise the possibility that Ir gene products may function in part at the level of the macrophage.     

Dissociation of fibronectin from gelatin-agarose by amino compounds.

Affinity chromatography of radioiodinated solubilized erythrocyte stroma and of radioiodinated purified Band 3 on an anti-(blood-group I)-adsorbent column showed blood-group-I activity associated with a subpopulation of Band 3. The specificity of binding was confirmed by inhibition with known blood-group substances in radioimmunoassays.     

Natural IgG antibody with anti-β-galactosyl specificity suppressed hepatoma cell invasion in culture

The effect of natural IgG antibody recognizing β-galactosyl epitope on hepatoma cell invasion was investigated. Anti-β-galactosyl antibody dose-dependently suppressed hepatoma invasion underneath primarily cultured mesothelial cells monolayer without affecting the proliferation, to the same extent as natural IgG antibody with anti-α-galactosyl specificity, which had already been reported to have an anti-metastatic activity. The inhibitory effect of anti-β-galactosyl antibody was completely canceled by adding lactose (galactose-β-1, 4-glucose) to the medium, indicating that this antibody recognized some antigens with β-galactosyl epitope. Hepatoma cells pretreated with this antibody for 48 h showed reduced invasive activity, while the pretreatment of mesothelial cells with the antibody did not affect hepatoma cells invasion. Anti-β-galactosyl antibody also suppressed hepatoma cells adhesion to mesothelial cells monolayer. These results suggest that natural antibody with anti-β-galactosyl specificity may recognize the β-galactosyl epitope in some adhesion-related molecules on hepatoma cells, thus suppressing adhesion and invasion to mesothelial cells monolayer. These results suggest possible therapeutic uses of this antibody in the treatment of metastatic tumors.     

Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites.     

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity change of antibody to (4-hydroxy-3-nitrophenyl)acetyl haptens by somatic hypermutation

Change in the specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (Abs) with time after immunization was studied. The early anti-NP Abs was specific to the ionized (phenolate) form of NP. The specificity changed with time and the late Abs became able to bind to the protonated (phenolic) form as well as the phenolate form of NP. The nucleotide sequences of mRNA coding for variable regions of heavy and light chains suggested that somatic hypermutation contributed to this change of the specificity.     

A biologically active fructan from the roots of Arctium lappa L., var. Herkules

From the roots of Arctium lappa L., var. Herkules a low-molecular-weight fructofuranan of the inulin-type has been isolated by water extraction and ethanol precipitation, followed by ion-exchange chromatography and gel filtration of the crude precipitate. The methods employed in structural determination were methylation analysis and 1H and 13C NMR spectral measurements. In tests for antitussive activity in cats the fructan was found to be equally active as some non-narcotic, synthetic preparations used in clinical practice to treat coughing, and in mitogenic and comitogenic tests its biological response was comparable to that of the commercial Zymosan immunomodulator.     

Localization of the metal-induced conformational transition of bovine prothrombin

The calcium-stabilized antigenic determinants on bovine prothrombin were localized to the NH2-terminal 1-42 residues using conformation-specific antibodies. Polyclonal antibodies to the bovine prothrombin-Ca(II) complex were raised in rabbits, and purified antibody subpopulations were isolated by sequential immunoabsorption and affinity chromatography. Anti-prothrombin-Ca(II) antibodies, characterized by their absolute specificity for the prothrombin-metal complex (Tai, M. M., Furie, B. C., and Furie, B. (1980) J. Biol. Chem. 255, 2790-2795), bound to prothrombin, fragment 1, reduced and carboxymethylated fragment 1, and CNBr fragment (1-72) in solution. However, these antibodies do not bind significantly to the gamma-carboxyglutamic acid-rich fragment (1-39), CNBr fragment (73-156), or prethrombin 1. To obviate the complex analysis of possible reasons for the lack of antibody binding to small peptides in solution, conformation-specific antibodies directed against defined regions of the whole prothrombin molecule were isolated. The influence of calcium ions on the binding of these site-specific antibody subpopulations to 125I-labeled prothrombin fragment 1 was evaluated. Anti-(1-39)N, anti-(1-42)N, anti-(1-72)N, and anti-(reduced and carboxymethylated fragment 1)N showed enhanced binding to prothrombin fragment 1 in the presence of Ca(II), indicating the presence of calcium-stabilized antigenic determinants within each of these regions on fragment 1. In contrast, calcium ions had no effect on the interaction of anti-des-(1-42)prothrombin, anti-prethrombin 1, anti-(43-72)N, and anti-(73-156)N antibodies with prothrombin fragment 1. These results indicate that the metal-induced conformational transition, monitored immunochemically, is localized to the NH2-terminal, gamma-carboxyglutamic acid-rich region of prothrombin between residues 1-42.     

Immunochemistry of scorpion toxins. Immunogenicity of peptide 19-28 a model of an accessible and relatively rigid region

Peptide 19-28, a model of an antigenic region of Androctonus australis Hector toxin II, has a rigid alpha-helix structure in the native protein. It was used as immunogen either in the free form, or bound to bovine serum albumin (BSA) or linked to its synthesis support (macroporous polyacrylamide resin). Only the anti-(peptide-BSA) and the anti-(peptide-resin) antibodies recognized the native toxin. The use of short synthetic analogues of peptide 19-28 suggests a specificity difference in the two antipeptides. Anti-(peptide-BSA) recognizes probably two determinants localized at the C and N terminals of the peptide chain. Anti-(peptide-resin) preferentially recognizes the N-terminal extremity. Finally we showed that the alpha-helix region remains accessible to antipeptide 19-28 when the toxin is bound to its receptor.     

Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA.

Autoantibodies exclusively precipitating U1 and U2 small nuclear ribonucleoprotein (snRNP) particles [anti-(U1,U2)RNP] were detected in sera from four patients with autoimmune disorders. When tested by immunoblotting, these sera recognized up to four different protein antigens in purified mixtures of U1-U6 RNP particles. With purified antibody fractions eluted from individual antigen bands on nitrocellulose blots, each anti-(U1,U2)RNP serum precipitated U2 RNP by virtue of the recognition of a U2 RNP-specific B" antigen (mol. wt. 28 500). Antibodies to the U2 RNP-specific A' protein (mol. wt. 31 000) were found in only one serum. The B" antigen differs slightly in mol. wt. from the U1-U6 RNA-associated B/B' antigens and can be separated from this doublet by two-dimensional gel electrophoresis, due to its more acidic pI. In immunoprecipitation assays, the purified anti-B" antibody specificity also reacts with U1 RNPs which is due to cross-reactivity of the antibody with the U1 RNA-specific A protein, as demonstrated by immunoblotting using proteins from isolated U1 RNPs as antigenic material. Thus the A antigen not only bears unique antigenic sites for anti-A antibodies contained in anti-(U1)RNP sera, it also shares epitopes with the U2 RNP-specific B" antigen.     

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis

Abstract We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti- Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.     

Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development

Concanavalin A and Robinia pseudoacacia lectin decreased [(3)H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6-10-day chick embryos. In contrast, these lectins stimulated [(3)H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [(3)H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with alpha-methyl mannopyranoside and with anti-(Robinia lectin) serum.     

Anti-ζ Antibody Screening for α-Thalassemia Using Dried Filter Paper Blood

The most common α-thalassemia in Southeast Asian or Southern Chinese populations is the (- -^SEA) double α-globin deletion. Couples heterozygous for (- - ^SEA) have 25% risk for hydrops fetalis from loss of all four α-globin genes. The (- -^SEA) deletion spares the embryonic ζ-globin genes and causes traces of ζ-peptide to persist throughout life. A colorimetric monoclonal anti-ζ antibody test for raised ζ-peptide has detected the (- -^SEA) deletion in liquid blood samples, but not deletions of the entire α-globin region with loss of the ζ-globin genes. Eluates from dried blood spots had the same anti-ζ antibody color reaction as whole blood, even after storage at 4°C for up to 77 days. The anti-ζ antibody test was positive in 24 of 91 microcytic samples (mean corpuscular hemoglobin <24 pg), including four with iron deficiency; it was negative in 26 provisionally diagnosed α-thalassemia-1 heterozygotes and all 32 nonmicrocytic samples. Southern blot analysis and a specific SEA-polymerase chain reaction test confirmed that 18 anti-ζ antibody-positive samples and 1 anti-ζ antibody-negative sample had the (- -^SEA) deletion. Two anti-ζ antibody-negative microcytic samples had the (- -^Fil) total α-globin region deletion, 2 had single α-gene deletions, 22 others may also have had a total α-region deletion. Hence specificity was very high and sensitivity was 95%. The anti-ζ antibody test can detect the (- -^SEA) deletion in dried blood samples, even after prolonged storage. This simple inexpensive test can conveniently screen samples collected at a distance from a central laboratory.     

Pyrolysis of inulin,glucose and fructose

The pyrolytic behavior of inulin, a (2 → 1)-linked fructofuranan, is described. Parallel investigations of the pyrolysis of glucose and of fructose were conducted to supplement the inulin results and to aid comparison with previous results from glucans. Effects of neutral and basic additives are emphasized. As with glucans, the addition of such additives (especially basic) increases the yields of the one-, two-, and three-carbon products (as well as of hexosaccharinolactones), while generally decreasing the yields of anhydro sugar and furan derivatives. The former products include glycoaldehyde, acetol, dihydroxyacetone, acetic acid, formic acid, and lactic acid. Mechanistic speculations are made regarding the origins of these compounds, as well as of furan derivatives and saccharinic acid lactones. Parallels with alkaline degradation are considered.