The physiological significance of the xanthurenic acid-insulin comples.

The xanthurenic acid-insulin complex was found to have similar immunological properties to native Zn-insulin. This complex showed less hormonal activity on glucose metabolism in adipose tissue than native An-insulin, but its activity was increased by addition of Zn2+ ions.     

Characterization of the type and group specificities of the immune response in mice to murine leukemia viruses.

Normal sera from a variety of strains of inbred mice have precipitating antibodies to murine type C viruses that are detected by radioimmune precipitation assays. The results demonostrate that this humoral immune response is primarily directed against the AKR strain of murine leukemia virus (MuLV) proteins gp71, gp43, and p15(E). These sera also react with Friend- or Rauscher-MuLV in radioimmune precipitation assays. This reaction is not due to a separate immune response, but rather is primarily a consequence of the cross-reactivity of antibodies to the AKR strain of MuLV p15(E) with the p15(E) of these viruses. These data, using autogenous immune sera, emphasize the serological differences of the virion glycoproteins and the serological similarity of the p15(E) virion component of the viruses. Furthermore, based on the serological reactivities to the glycoproteins, the results suggest that the AKR strain of MuLV is endogenous to and expressed in mice, but that the Friend-Moloney-Rauscher virus group is not.     

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.     

The reactivity of lupus erythematosus serum with native and denatured DNA of various origins]

The serum of a female suffering from Lupus erythematosus visceralis was investigated by complement fixation for the reaction with native and denatured DNA's of various base composition. The reaction with native DNA is independent on the (G+C)-content of the DNA. It is apparent that the responsible antibodies react with determinants of the helical conformation, which are identical in the various DNA-molecules. Quantitative differences are found with denatured DNA's. The strongest complement fixation is observed with (G+C)-rich denatured DNA. The reaction with denatured DNA is only partially inhibited by DNA digest. These antibodies obviously react with sequential determinants containing bases. Therefore, they are induced by a different mechanism of sensitization.     

Purification and serological characterization of the major envelope glycoprotein from AKR murine leukemia virus and its reactivity with autogenous immune sera from mice.

The major envelope glycoprotein (gp71) from AKR murine leukemia virus (MuLV) was purified and its serological reactivity with heterologous and autogenous immune mouse sera was examined. Homologous and interspecies competition radioimmunoassays using antisera to Rauscher-MulV gp69/71 or Friend-MuLV gp71 or antisera to feline leukemia virus to precipitate 125I-labeled gp71 from various MuLV showed that distinct differences exist between Rauscher- or Friend-MuLV and AKR-MuLV glycoproteins. Characteristically the AKR-MuLV gp71, in contrast to FLV or RLV gp71, does not compete fully in homologous or interspecies radioimmunoassays with iodinated Friend of Rauscher glycoproteins. Purified 125I-labeled AKR-MuLV gp71, in contrast to the Rauscher- or Friend-MuLV glycoproteins, reacts with normal (autogenous immune) mouse sera in direct radioimmune precipitation assays. Competition experiments further demonstrate that this is a predominant immunological reactivity of normal mouse sera which had previously been detected by radioimmune precipitation assay against intact virions.     

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.     

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.     

Anti-idiotype antibodies prevent antibody binding to mouse sperm and antibody-mediated inhibition of fertilization

Antibodies to components of sperm can interfere with sperm function and prevent fertilization by blocking specific steps of gamete interaction. It can be proposed that anti-idiotype antibodies (anti-Ids) that recognize determinants located close to or within the antigen-binding site of an anti-sperm antibody could block antibody binding to sperm antigen and antibody-mediated inhibition of fertilization. To test this hypothesis, rabbit polyclonal antibodies to idiotypic determinants of the monoclonal anti-sperm antibody M42.15 were developed and characterized. Previous studies demonstrated that M42.15 monoclonal antibody (mAb) inhibits fertilization in vitro and in vivo by inhibiting sperm-zona pellucida interaction. Anti-idiotype antibodies to M42.15 mAb (anti-Id M42) were isolated by affinity chromatography on immobilized M42.15 mAb. Binding specificity of anti-Id M42.15 was demonstrated in a solid-phase radioimmune binding assay and by specific immunoprecipitation of soluble M42.15 mAb. Anti-Id M42 competitively inhibited M42.15 mAb, but not P220.2 mAb, binding to mouse sperm, confirming that the anti-Id preparation contained antibodies directed against idiotopes within or adjacent to the antigen-binding site of the mAb. At equimolar concentrations, anti-Id M42 inhibited binding of 125I-labeled M42.15 mAb to sperm by greater than 80%. These results showed that anti-Id M42 efficiently blocked antibody binding to sperm and suggested that anti-Id M42 could be used to neutralize the anti-fertility activity of the M42.15 mAb. When tested in in vitro fertilization assays, anti-Id M42, but not rabbit immunoglobulin, prevented M42.15 mAb-induced inhibition of fertilization. Together, these results show that the inhibitory activity of anti-sperm antibodies capable of interfering with gamete interaction can be neutralized by anti-Id that recognize determinants close to the antigen-combining site of the anti-sperm antibody.     

The human immune response to insulin. I. Kinetic and cellular aspects of lymphocyte proliferative responses in diabetics

A reproducible culture system was developed with the use of peripheral blood mononuclear cells (MNC) from insulin-dependent diabetic subjects to assay T cell proliferation in response to mammalian insulins. Kinetic analysis revealed that maximal responses occur after 8 to 10 days of culture with 50 to 100 micrograms of insulin. Characterization of the cells involved showed that two cell types are required for this proliferative response: a radioresistant non-T cell and an E rosette-forming (T) lymphocyte. With the use of this assay, 16 of 32 insulin-dependent diabetics had demonstrable MNC proliferation in response to insulin or to the control antigen, tetanus toxoid. Among those 16 subjects a spectrum of responsiveness to insulin was found. Because MNC from all responsive subjects react to both beef and pork insulin, it appears that shared determinants are recognized by insulin-immune lymphocytes in this outbred population. In addition, cells from one subject were found to respond in a determinant-specific manner to the A-chain loop of beef insulin. Reproducibility of the assay was demonstrated over an 8-mo period in three experiments with the use of MNC from a single subject. This reliable assay of T lymphocyte responses to a defined antigen will help determine the fine specificity and genetic regulation of the immune response to insulin in man.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Extracellular Amastigote-Like Forms of Leishmania panamensis and L. braziliensis. II. Stage- and Species-Specific Monoclonal Antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania . Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to >200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Chondroitin 6-sulfate oligosaccharides as immunological determinants of chick proteoglycans

Monoclonal antibodies to hyaluronidase-treated chondroitin sulfate proteoglycan (CSPG) were used to study the immunological determinants of chick cartilage proteoglycan. The determinants recognized by the antibodies were studied by a radioimmune inhibition assay utilizing hyaluronidase-treated [35S]CSPG. Hyaluronidase-treated CSPG inhibits the reaction of four clonal antibodies, S54C, S103L, S11D, and P100D, with [35S]CSPG, but to varying degrees. Only the reaction of S103L is inhibited to a considerable extent by undigested CSPG, indicating that hyaluronidase treatment exposes determinants specific for the other three antibodies. These findings are consistent with the earlier conclusion that S103L is specific for a protein determinant (Dorfman et al., 1980). Only the reaction of S54C is not significantly inhibited by chondroitinase ABC-digested CSPG. This result indicates that chondroitinase ABC digestion can also expose determinants recognized by S11D and P100D but that such digestion removes the determinant recognized by S54C. Of the four antibodies tested, only the reaction of S54C with hyaluronidase-treated [35S]CSPG is significantly inhibited by chondroitin-6-SO4 tetra- and hexasaccharide (59 and 43% inhibition, respectively, at a concentration of 1333 microM). The reaction of S54C is inhibited to a lesser extent by chondroitin tetra- and hexasaccharide (28 and 26% inhibition, respectively, at a concentration of 1333 microM). In contrast, chondroitin-4-SO4 oligosaccharides do not inhibit the reactions of any of the clonal antibodies. These result suggest that S54C recognizes a determinant that contains chondroitin-6-SO4 oligosaccharide, attached via the linkage oligosaccharide to core protein.     

Metal induced conformational changes in human insulin: crystal structures of Sr2+, Ni2+ and Cu2+ complexes of human insulin

Crystal structures of Sr(2+), Ni(2+) and Cu(2+) of human insulin complexes have been determined. The structures of Sr(2+) and Ni(2+) complexes are similar to Zn(2+) insulin and are in T6 conformation. (All the six monomers in the insulin hexamer are in Tensed conformation (T), which means the first eight residues of B-chain are in an extended conformation). Cu(2+) complex, though it assumes T6 conformation, has more structural differences due to lowering of crystal symmetry and space group shift from H3 (Hexagonal crystal system) to P3 (Trigonal crystal system) and a doubling of the c axis. 2Ni(2+) human insulin when compared to 4Ni(2+) Arg insulin suggests that terminal modifications may be responsible for additional metal binding. All the three metals have been shown to have a role in diabetes and hence may be therapeutically useful.     

Inhibition of the activation and catalytic activity of insulin receptor kinase by zinc and other divalent metal ions

The autophosphorylation reaction responsible for conversion of insulin receptor (from human placenta) to an active tyrosyl-protein kinase was shown to be inhibited by Zn2+ and other divalent metal ions. The order of inhibitory potency was found to be Cu2+ greater than Zn2+, Cd2+ greater than Co2+, Ni2+. Autophosphorylation of insulin receptor was almost completely blocked by 10 microM Zn2+. Zn2+, however, did not appear to affect the binding of insulin to its receptor. Histidine, a chelator of Zn2+, protected against the inhibitory effects of Zn2+. The failure of histidine to regenerate the competence of the Zn2+-inhibited receptor to undergo autophosphorylation suggested that the inhibition by Zn2+ was irreversible. In addition to inhibiting autophosphorylation, Zn2+ inhibited the tyrosyl-protein kinase activity of highly purified phosphorylated receptor. Zn2+ was also observed to inhibit phosphotyrosyl-protein phosphatase activity present in preparations of partially purified insulin receptor. These inhibitory effects of Zn2+ should be considered in the design of protocols for the isolation and handling of insulin receptor and possibly other tyrosine kinases. Additionally, the possible physiological significance of the inhibition of insulin receptor kinase by Zn2+ is discussed in light of the fact that Zn2+ is accumulated in and secreted from pancreatic islet cells together with insulin.     

Structural and antigenic differences between two types of meningococcal pili

Abstract Strains of meningococci, which were shown to be pilated by electron microscopy, could be divided into two groups on the basis of antigenicity and subunit M _r. Strains from group 1 which reacted with monoclonal antibodies directed against gonococcal pili, had pili with subunit M _r similar to that of gonococci which could be detected by radioimmune precipitation or electroblotting. Strains from group 2 failed to react with the monoclonal antibodies and had pili with lower subunit M _r which could only be detected by radioimmune precipitation using polyclonal antipilus antiserum and not by electroblotting.     

Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.     

Separation of neutralizing and hemagglutination-inhibiting antibody activities and specificity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions.

Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived polyoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying 125I-labeled immunoglobulins to SDS-polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacrylamide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutralizing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immunoprecipitation experiments to demonstrate that the antigenic determinants responsible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both of these polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1.     

Monoclonal antibodies to the glucose transporter from human erythrocytes. Identification of the transporter as a Mr = 55,000 protein

Using the preparation of purified glucose transporter from human erythrocytes as antigen, we have prepared and characterized six monoclonal antibodies. Three of these antibodies have been shown to be to the glucose transporter by several criteria: they immunoprecipitate the transport activity, the cytochalasin B binding activity, and 75% of the protein from the solubilized purified preparation. The remaining three antibodies were shown to recognize the same polypeptide by a Western blot procedure. All of the antibodies reacted with the deglycosylated transporter and are thus against peptide determinants; most bound to the cytoplasmic domain of the transporter. The antibodies exhibited a range of effects on cytochalasin B binding, from slight enhancement to modest inhibition to strong inhibition; for this reason they must bind to at least three different epitopes. Western blot analysis of erythrocyte membranes prepared in the presence of protease inhibitors showed that all six antibodies bound to a polypeptide of average Mr = 55,000. Moreover, by immunological assay this polypeptide accounted for 5.3% of the membranes protein, a value similar to that given by cytochalasin B binding. Thus, the proposal that the native transporter is a Mr = 100,000 polypeptide is highly unlikely. The antibodies also react with the glucose transporter in other human cell types, but not with that in rodent or avian cells.     

Subspecies- and species-specific antigens of Leishmania mexicana characterized by monoclonal antibodies

Monoclonal antibodies have been produced that are specific for the reference stocks of Leishmania mexicana species and subspecies L. mexicana mexicana(L11, M379), L. mexicana amazonensis (WR303, H6, LV72), and L. mexicana pifanoi (L20). The specificities of these antibodies were confirmed by analyses employing an indirect radioimmune binding assay and 107 stocks of New World Leishmania. The molecules associated with these species- and subspecies-specific determinants have been characterized by Western blot analysis and consist of mainly low m.w. (11,000 to 50,000) membrane-associated components.     

Cell surface T antigen in cells infected with simian virus 40 or an adenovirus-simian virus 40 hybrid, Ad2+D2.

Cell surface T antigen, detected by a radioimmune assay that uses 125I-labeled Staphylococcus aureus protein A and antibodies against either authentic T antigen or D2 hybrid T antigen, was found in simian virus 40-transformed and -infected cells and in cells infected with an adenovirus-simian virus 40 hybrid, Ad2+D2. In simian virus 40 lytic infection, the surface T antigen appeared at the same time as the nuclear T antigen.