Immunochemical characterization of esterase active antigens in rat liver microsomes.

Ten distinct immunoprecipitates with esterase activity were identified in the reaction between rat liver microsomes and rabbit anti-rat liver microsomal antisera in crossed immunoelectrophoresis. These esterases were further characterized by the use of inhibitors, different substrates, hormone induction experiments and cross-reactivity studies with microsomes from other organs.Two of the esterase active antigens were found to be present in 2–3-fold higher concentrations in microsomes from female than male liver. Administration of the female sex hormone estradiol to male rats caused an induction of these esterases to the female level.The microsomal esterases showed a preference to hydrolyze substrates with short fatty-acid chains. Only one esterase could attack acetyl- and butyrylthiocholine. It was also inhibited by 10^?3M eserine and was thus considered to be a cholinesterase.All esterases except one were inhibited by 10^?3M DFP. In cross-reactivity experiments it was shown that this DFP insensitive esterase was also present in lung and testes microsomes. Kidney microsomes had two other esterases in common with liver, while microsomes from intestine, ovaries and heart gave one weak and diffuse precipitate, not further characterized, with the rabbit anti-rat liver microsomal antiserum.     

Immunochemical studies of microsomal membranes of rat preneoplastic and neoplastic hepatocytes

Heterogenous rabbit antisera were prepared against microsomal proteins of hyperplastic hepatic nodules (HPN) induced by chemicals, and were utilized to assess the antigenic differences of microsomal polypeptides within a normal liver, HPN, and hepatocellular carcinomas (HCC), utilizing immunodetection of antigens separated electrophoretically and transferred to nitrocellulose. Although most antigens were common to all microsomes, differences (increase or decrease) were noted in some polypeptides not only between the normal liver and HPN, but also between HPN and HCC. On the other hand, monoclonal antibodies against epoxide hydrolase (EH), which was initially found as the PN antigen, reacted to a single polypeptide with a molecular weight of 49,000 in all the microsomes. These results suggested that there is little molecular modification of EH during hepatic carcinogenesis.     

Immunochemical characterization of platelet-specific alloantigen Baka

We investigated the location of platelet-specific alloantigen Baka on platelet membrane glycoproteins. In indirect immunoprecipitation experiments, the anti-Baka antibody precipitated glycoprotein (GP) II b and a small amount of GP III a. The immunoblots using partially purified GP II b/III a complex as the target antigen indicated that GP II b alpha carried the Baka alloantigen. When the partially purified GP II b/III a complex digested with chymotrypsin was examined, the Baka alloantigen was found on a 65 kD fragment derived from GP II b alpha under reducing conditions. In addition, the immunoblots after two-dimensional nonreduced-reduced SDS-PAGE directly indicated that the 65 kD fragment had a mol. wt. of 80 kD under nonreducing conditions. The immunoblots using platelets digested in situ with chymotrypsin indicated that the 65 kD fragment of GP II b alpha was retained by the platelet membrane. We conclude, therefore, that the Baka alloantigen is located on a 65 kD fragment that represents the membrane side of the cleavage site of chymotrypsin on GP II b alpha.     

Immunochemical characterization of rough Brucella lipopolysaccharides

Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its reaction of partial identity in immunodiffusion. Monospecific mouse sera against B. ovis R-LPS agglutinated only the homologous bacteria but not R cells of other species of Brucella. B. ovis R-LPS contained more 2-keto, 3-deoxyoctonate, and glucosamine as a percentage of dry weight than any other R-LPS tested. B. abortus R-LPS was identified by the absence of an unidentified sugar present in the other R-LPS molecules, and B. melitensis R-LPS could be differentiated from B. canis R-LPS by its higher content of fatty acids. In contrast to S-LPS, all of the R-LPS studied lacked quinovosamine. In electron micrographs, Brucella R-LPS had a granular appearance, in contrast to typical lamellar structures formed by Brucella S-LPS and Escherichia coli R-LPS.     

Immunochemical characterization of cocos nucifera pollen

The Cocos nucifera pollen, as one of the sources of allergen responsible for immediate hypersensitivity reaction, was confirmed by skin prick test, bronchial provocation test, and RAST. The whole pollen extract (WPE) of C. nucifera was fractionated by combination of gel filtration and ion-exchange columns with fast protein liquid chromatography (Pharmacia, Uppsala, Sweden). Three protein peaks designated Cocos II, Cocos VI, and Cocos VII exhibited allergenic properties, as tested by skin prick test, direct IgE ELISA, bronchial provocation test, and immunoblot analysis. In RAST inhibition, Cocos IIa (a high-molecular-weight protein) obtained by fractionation of Cocos II on Mono Q column (fast protein liquid chromatography) (Pharmacia) was found to be the most potent allergen in Cocos WPE, followed by Cocos VI and Cocos VII, which are low-molecular-weight proteins. The reference patterns of Cocos WPE on crossed immunoelectrophoresis and thin-layer isoelectric focusing were established for future standardization of Cocos WPE to be used in the diagnosis and immunotherapy of allergic patients.     

Ionic fluxes and permeabilities of cell membranes in rat liver

1. Intracellular ion concentrations, measured in rat liver perfused with saline solutions were, at steady state:[K](1) = 113; [Na](1) = 16.4; [Cl](1) = 25.5 m-mole l.(-1) of cells.2. Intracellular Cl concentration was measured when both [Cl](o) and membrane potential were changed. The experimental values were close to the predicted ones by the Nernst equation, indicating a passive distribution of this ion across the cell membrane.3. Fluxes were determined by means of radioactive tracers and had the following values:m(K) = 6.6; m(Na) = 12.4 and m(Cl) = 8 x 10(-12) mole cm(-2) sec(-1).4. When Na was replaced by Li in the perfusing solutions, the Na efflux was decreased by 3.3 x 10(-12) mole cm(-2) sec(-1). This was attributed to a Na-for-Na exchange (exchange-diffusion).5. A mathematical model was applied to the perfused liver. It allowed estimation of the actual fluxes across the membrane. Corrections resulting from the application of the model remain small.6. The permeability coefficients were calculated from the passive fluxes and were:P(K) = 7.6; P(Na) = 4.0; P(Cl) = 12.3 x 10(-8) cm sec(-1), corresponding to relative permeabilities of P(Na)/P(K) = 0.52 and P(Cl)/P(K) = 1.6.7. The membrane potential calculated from the Goldman equation was significantly different from the measured one. This may be accounted for by an electrogenic activity of the Na-K pump. Applying the Mullins & Noda equation, the ratio of active Na flux to active K flux becomes 3/2.     

Immunochemical characterization of edible bird's nest allergens

BACKGROUND: We have previously described anaphylaxis induced by edible bird's nest (BN) and demonstrated that this condition is IgE mediated. OBJECTIVES: This study aimed at describing the immunochemical properties of the BN allergens. Comparative studies between 3 commercially available sources (according to the country of origin) of BN were also made. METHODS: Crude extracts of commercially available processed BN from Sarawak (Malaysia), Thailand, and Indonesia and fresh unprocessed BN from the caves of Sarawak were obtained by means of aqueous extraction. Specific IgE toward these sources were determined by using fluorescence allergosorbent tests (FASTs). Cross-reactivity studies between the 3 sources of commercially available processed BN were carried out by means of FAST inhibition. Immunochemical characterization by means of IgE immunoblot, periodate treatment, and heat stability studies were carried out on fresh unprocessed BN from Sarawak. RESULTS: Serum from allergic patients showed differences in IgE binding to the 3 sources of commercially available BN, with the highest levels of specific IgE recorded with the Sarawak source (P <.0001). Of these, only the Sarawak and Thailand sources showed considerable cross-reactivity. Further work on the unprocessed fresh Sarawak source identified a putative 66-kd major allergen containing several isoforms. Periodate treatment resulted in loss of IgE binding. Despite a progressive decline in the molecular weights of allergens on SDS-PAGE with increasing periods of boiling, IgE binding, as assessed by means of FAST, was not affected. N-terminal sequence of the major putative allergen (66 kd) showed homology to a domain of an ovoinhibitor precursor in chicken (SWISS-PROT accession No. P10184). CONCLUSIONS: We have described the immunochemical properties of BN allergens. Edible BN from different sources are allergenically dissimilar. The putative major allergen is a 66-kd protein.     

Immunochemical characterization of Aspergillus fumigatus antigens.

Culture filtrate antigens of Aspergillus fumigatus Ag 534 were purified by preparative isoelectric focusing and affinity chromatography. One of the pooled antigen fractions from the preparative isoelectric focusing step (pool 2) was passed through a concanavalin A column and yielded two components, designated antigens IIa and IIb. Antigen IIb reacted more strongly than antigen IIa with all of the aspergilloma and allergic bronchopulmonary aspergillosis sera tested by enzyme-linked immunosorbent assay. The glycoprotein nature of antigen IIb was shown by the concanavalin A binding properties and staining reactions of the components.     

Phospholipid modifications and adenylate cyclase in rat liver plasma membranes

Rat liver plasma membrane phospholipid headgroups or fatty acids were modified by enzymatic transmethylation or transacylation. To evaluate the effect of phospholipid modifications on adenylate cyclase (AC), one of the enzymes of phospholipid metabolism as well as AC were measured in the same incubation medium. Methyl transfer fromS-adenosyl-l-methionine (SAM) to phosphatidylcholine (PC) was the highest at pH 9.2. At low concentrations of SAM, synthesis of PC as well as synthesis of the monomethyl derivative were considerably decreased and Mg²⁺ had no effect at pH 9.2 or at pH 6.5. When phospholipid methylation was increased in relation to SAM concentration, there was no change in basal, NaF- and glucagon-stimulated AC. Methylation was not modified when AC was stimulated. On the other hand, there was an increase in the basal, NaF- and glucagon-stimulated AC when linoleate was incorporated into the membrane phospholipids. Other unsaturated fatty acids had no effect. The synthesis of linoleoyl-PC from added lysophosphatidylcholine (LPC) also stimulated AC and this in turn partly prevented the inhibitory effect of LPC. Thus in isolated plasma membranes transmethylation has no direct effect on AC, whereas synthesis of linoleoyl molecular species can modulate AC in a way which does not depend on the membrane fluidity.     

Immunochemical characterization of brain and pineal tryptophan hydroxylase

Recombinant mouse tryptophan hydroxylase (TPH) was expressed in Escherichia coli, using a bacterial expression vector and has been purified to homogeneity by sonication followed by Sepharose 4B column chromatography and native slab gel electrophoresis. This purified enzymatically active TPH protein was used for production of a specific antiserum. This antiserum identified the predicted TPH band (molecular weight, 54 kDa) on Western blot of crude extracts from the rat and mouse dorsal raphe, and the rat pineal gland. However, this antiserum recognized an additional protein band of lower molecular weight (48 kDa) in pineal extract. It is not clear whether the 48 kDa TPH band represents an isozyme or a protease cleavage product of TPH. Since the pineal gland contains higher TPH mRNA and lower TPH activity when it is compared with dorsal raphe nucleus enzyme, this lower molecular weight TPH may participate in the reduced TPH specific activity. In addition, there are no specific TPH inhibitors in the pineal gland and this lower molecular weight TPH is inactive or has a very low specific activity. This antiserum specifically immunostained serotonergic cell bodies in the dorsal raphe nuclei, some large caliber serotonergic processes in the dorsal raphe area as well as terminals in the olfactory bulb. It also immunolabeled the pineal gland and immunoprecipitated equally well TPH protein from the dorsal raphe nucleus and the pineal gland in a concentration-dependent manner.     

Immunochemical characterization of Brucella lipopolysaccharides and polysaccharides.

Purified lipopolysaccharide (LPS) extracted with phenol-water from smooth Brucella abortus was hydrolyzed with 1% acetic acid at 100 degrees C. The degraded polysaccharide (AH) released gave reactions of identity with the native polysaccharide hapten (NH) in phenol-water- or trichloroacetic acid-extracted endotoxin preparations of B. abortus and with the polysaccharide (poly B) extracted by trichloroacetic acid from rough B. melitensis strain B115. Poly B was present in the soluble cytoplasmic fraction but not in the membrane fraction, of disrupted B115 cells. It could not be extracted from three rough mutants of B. abortus or from B canis or B. ovis cells. Both AH and NH shared determinants present on smooth LPS and missing from poly B. Sugars found in purified LPS, NH, and AH included mannose, glucose, quinovosamine, glucosamine, and 2-keto-3-deoxyoctonate. Poly B contained only a trace amount of quinovosamine and no 2-keto-3-deoxyoctonate detectable by the thiobarbiturate assay. Sera from some rabbits immunized with pure smooth LPS and some, but not all, cows infected with field strains of B. abortus recognized the determinants missing from poly B. A subclass-specific enzyme-linked immunoassay showed that most of the antibody in sera from infected cows which binds to smooth LPS and to NH is of the immunoglobulin G1 subclass.     

Immunochemical characterization of human antibodies to lymphoblastoid interferon.

Antibodies produced in recurrent respiratory papillomatosis (RRP) patients treated with lymphoblastoid interferon (lyIFN) may neutralize antiviral activity or may only bind to lyIFN. These antibodies were characterized for immunoglobulin class, IgG subclass, and light chain type by an indirect immunoassay. Serum dilutions were incubated on lyIFN-coated plates and the presence of antibody detected using peroxidase-conjugated goat antibodies to each human immunoglobulin class and light chain isotype, or using MoAbs to each human IgG subclass. Neutralizing activity was measured as the inhibition of lyIFN antiviral activity for Vervet monkey cells challenged with Semliki Forest Virus. Among antibody-positive patients, 12% produced IgM coincident with IgG, and 25% produced IgA coincident with IgG. Thus, antibody responses in patients treated with lyIFN are not exclusively of IgG class. The predominant lyIFN-specific subclasses were IgG1 and IgG3, which occurred in 70% and 83% of patients, respectively. An IgG4 response was detected in two patients who also had antibody of other isotypes; no IgG2 antibody was detected in any patient. Antibodies were not IgG subclass-restricted, a trend which was more pronounced in patients having neutralizing antibody than non-neutralizing antibody. Light chain molecules of lyIFN-specific antibody were of both kappa and lambda isotypes, with kappa chains occurring most frequently. Among patients having non-neutralizing antibodies, monotypic light chains occurred in 65% of the patients, whereas no patient with neutralizing antibody had monotypic light chain antibody. Sera from 599 normal human volunteers were assayed for antibody, and seven were found to be immunoreactive to lyIFN. Only one serum of the seven was positive for neutralizing activity.     

Immunochemical study of liver metastases of colonic tumors]

This work demonstrated that anti-CEA and anti-NCA antibodies can be obtained from liver metastases of colonic carcinoma. The various antigens that have been identifiedin colonic tumor are found in liver metastases. Furthermore, the healthy parts of a metastatic liver contain not only CEA but also NCA.