Synthetic peptides from region 65–84 of bovine myelin basic protein: Radioimmunoassays and equilibrium competitive inhibition studies with antibodies prepared against myelin basic protein

Synthetic peptides with various and overlapping sequences represented by the residue region 65–84 of bovine myelin basic protein (MBP-bov) were tested in sodium sulfate radioimmunoassays for their reactivity with 15 rabbit antisera against MBPs from six different animal species and nine pools of syngeneic Lewis rat anti-MBP antisera. Three of the peptides were labeled with¹²⁵I and studied by direct binding reactions: (1) the prototype peptide S82 sequence TTHYG-SLPQKAQGHRPQDEG, corresponding to residues 65–84 of bovine MBP except for the C-terminal glycine, which is encephalitogenic in rabbits: (2) S81, which lacks the first three residues of S82 and is nonencephalitogenic in rabbits; and (3) S79, which represents the C-terminal half of S82 and which, because of its C-terminal glycine instead of asparagine, is nonencephalitogenic in Lewis rats. Fourteen of the rabbit anti-MBP antisera were reactive with [¹²⁵I]S82 (two borderline) including 2 of 3 antisera against human MBP, one against monkey MBP, and one against chicken MBP. The cross-reactions with [¹²⁵I]S81 were somewhat fewer and less intense. There were no cross-reactions with [¹²⁵I]S79. None of nine different pools of syngeneic rat MBP antisera cross-reacted with any of the three labeled peptides. With the use of a rabbit anti-MBP-rat antiserum that crossreacted strongly with [¹²⁵I]S82, 15 additional peptides with overlapping sequences within the residue region 65–84, as well as five MBP preparations from four different species, were tested by equilibrium and nonequilibrium competitive inhibition RIAs. Unlabeled S82 and MBP-bov were completely competitive with [¹²⁵I]S82 in the equilibrium assays; S81 and three other peptides had low degrees of cross-reactivity; but none of the remaining eight unlabeled peptides or unlabeled MBP preparations of guinea pig, rat, or mouse origin gave any evidence of competitive activity. Nonequilibrium competitive inhibition RIAs, however, did reveal cross-reactivities among several of the peptides as well as guinea pig and rat MBP. It was concluded that the N-terminal half of S82, particularly residues 68–74 (YGSLPQK), must contain an immunodeterminant of amino acid residues which identifies with the corresponding and exposed sequence in intact MBP-bov.This research was supported at Duke University by research grants 833-E-5 from the National Multiple Sclerosis Society and NS-10237 from the National Institutes of Health of the U.S. Public Health Service; at St. Luke's Hospital Center and Columbia University by grant RG1197-A-5 from the National Multiple Sclerosis Society; and at Northwestern University by grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

Immunochemical analysis of Lewis rat antisera to the synthetic encephalitogenic peptide S49

Discrete populations of anti-S49 antibodies were found in the antisera of Lewis rats recovered from S49-induced experimental allergic encephalomyelitis (EAE). A potent inducer of EAE in Lewis rats, S49 is a synthetic peptide representing residues 69–84 of bovine myelin basic protein but with deletions at Gly-77 and His-78 to form an analogue of guinea pig or rat 69–84, GSLPQKAQRPQDENG. Each population within a given antiserum, as identified by Scatchard and Sipsian window analysis, was found to exhibit reactivity for a different S49 determinant, and the affinities of each population were relatively restricted and discontinuous. The high affinity populations (10⁷–10⁸ M^–1) were cross-reactive with YS8 (YGSLPQKAQGHRPQDENG) in equilibrium competitive inhibition reactions whereas the low affinity populations (10⁵–10⁶ M^–1) were reactive only with S49 and YS49 among a panel of peptide analogues. Of the YS8 cross-reactive antibodies the highest affinity (10⁸ M^–1) were also cross reactive with S81 (YGSLPQKAQGHRPQDEG) but not S49 (69-84-Gly), thus emphasizing the need for Tyr-68 for format stability of the determinant involved. The other YS8 cross-reactive population (10⁷ M^–1) was completely reactive with S49 but totally unreactive with S81 in equilibrium reactions, thus emphasizing the requirement for Asn-84 but not Tyr-68 for the determinant's topographic stability. Peptides shorter than S49 from the N-terminal end, but retaining the sequences AQRPQDEN or SQRSQDEN (suspected residence of minimal encephalitogenic determinants), reacted only under conditions of two-step non-equilibrium competitive inhibition assays. Such reactions would occur only at very low affinity (<10⁵ M^–1) with the anti-S49 antibodies. It was hypothesized that the encephalitogenic T-cell determinant for Lewis rats, although permitting B-cell responses at very low affinity, may exclude high affinity responses in susceptible animals.This work supported at Duke University Medical Center by research grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service, Immunology Training Grant #5-T32-CA-09058-10, and Medical Scientist Training Program #5-T32-OM-07171-08, and at St. Luke's Hospital Center by NS-21466 from the National Institutes of Health and RG1197-B7 from the National Multiple Sclerosis Society.     

Affinity purification of two populations of antibodies against format determinants of synthetic myelin basic protein peptide S82 from S82-AH- and S82-CH-Sepharose 4B columns

Two different kinds of immunosorbents were prepared that contained the synthetic myelin basic protein didecapeptide S82 (TTHYGSLPQKAQGHRDQDEG)—one coupled with AH-Sepharose 4B through hexanoate spacers to the C-terminal glycyl residue; the other, with CH-Sepharose 4B through hexanoate spacers to the N-terminal threonine residue. An antiserum rich in antibodies to a format determinant of S82 was passed through each column, and, by means of affinity purification, two homogeneous populations of anti-format antibodies were obtained, each with a binding affinity of 1×10⁸M^–1 for S82. The population recovered from S82-AH-Sepharose 4B cross-reacted to a considerable extent with synthetic peptide S8 (GSLPQKAQGHRPQDENG) but only to a limited extent with S79 (AQGHRPQDEG). The population recovered from S82-CH-Sepharose 4B crossreacted poorly, if at all, with S8. An equimoler mixture of S8+S79, however, reacted well with either population of anti-format antibodies, thus showing that the mixture could mimic the format of S82. It was concluded that secondary structural conformation of S82 could be preserved during the coupling procedure and that the resulting immunosorbents could be used for the affinity purification of anti-S82 antibodies to the format determinants.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.Supported by Research Grants NS-10237 (Duke) and NS-15322 (St. Luke's) from the National Institutes of Health and by RG1197-B7 from the National Multiple Sclerosis Society.     

Format determinants of synthetic myelin basic protein peptide S82 mimicked by a mixture of synthetic peptides S8 and S79

Antibodies to synthetic myelin basic protein peptide S82 (TTHYG-SLPQKAQGHRPQDEG) did not react with synthetic peptide S8 (GSLPQKAQGHRPQDENG) and only partially so with synthetic peptide S79 (AQGHRPQDEG); however, the antibodies did react to a considerable extent with an equimolar mixture of S8 and S79. Since the anti-S82 antibodies had previously been shown to be directed to a non-sequential format determinant dependent on the conformation of secondary structure, it seems probable that the mixture of S8 and S79 assumed a format that neither one individually possessed to any great degree.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.Supported by Research Grants NS-10237 (Duke) and NS-15322 (St. Luke's) from the National Institutes of Health and by RG1197-B7 from the National Multiple Sclerosis Society.     

Evidence for a new subclass of calcitonin/ calcitonin gene-related peptide binding site in rat brain

Binding sites for calcitonin and calcitonin gene-related peptide are widely distributed in the central nervous system. In this study, binding of [¹²⁵I]-alpha-rat calcitonin gene-related peptide and [¹²⁵I]-salmon calcitonin in adjacent sections of rat brain revealed clearly distinct patterns of binding in most regions although in some restricted areas such as parts of the ventral striatum, including the nucleus accumbens, there was some overlap in the patterns of binding. In the primary olfactory cortex, which bound only calcitonin gene-related peptide, salmon calcitonin was very weak in inhibiting the binding of calcitonin gene-related peptide. In the nucleus accumbens, high affinity binding of calcitonin and calcitonin gene-related peptide at their homologous receptors was observed, with affinity constants for calcitonin and calcitonin gene-related peptide of 1.4 × 10⁹ M⁻¹ and 1.2 × 10⁹ M⁻¹ respectively. Cross competition studies in this nucleus demonstrated that salmon calcitonin was able to compete for [¹²⁵I]-rat calcitonin gene-related peptide labelled sites with high affinity, with an affinity constant of 0.8 × 10⁹ M⁻¹. However, rat calcitonin gene-related peptide was less potent in inhibiting the binding of [¹²⁵I]-salmon calcitonin labelled sites with only 28% inhibition at 10⁻⁶M. Further characterization of the calcitonin sensitive calcitonin gene-related peptide labelled sites demonstrated that a range of calcitonin analogs inhibited the binding of [¹²⁵I]-rat calcitonin gene-related peptide with the same order of potency as the analogs competed for [¹²⁵I]-salmon calcitonin labelled sites. Digital substraction mapping revealed calcitonin-sensitive calcitonin gene-related peptide binding sites over parts of the ventral striatum, including mid-caudal nucleus accumbens and fundus striati; over the lateral border of the lateral bed nucleus of the stria terminalis; part of the central amygdaloid nucleus; the organum vasculosum of the lamina terminalis and area postrema and over the wings of the dorsal raphe.These results demonstrate the existence of a new subtype of calcitonin/calcitonin gene-related peptide binding site, which has high affinity for the two otherwise biochemically distinct peptides.     

A rabbit B cell determinant for a conserved portion of myelin basic protein, rabbit encephalitogenic sequence 65-74

Synthetic peptide S24 (TTHYGSLPQKG) represents residues 65-74 of myelin basic protein (MBP) and contains the major determinant involved in the development of experimental allergic encephalomyelitis (EAE) in rabbits. This peptide is completely conserved in all nonprimate mammals for which sequence information is available. Although it is clear that peptides containing the S24 region are capable of inducing EAE, previous serologic studies have resulted in the conclusion that the determinant is "buried" or sequestered in intact MBP. Employing a liquid phase radioimmunoassay, we studied Ab responses to the S24 determinant in six rabbits injected with rat myelin. Two of the six animals developed small but measurable responses to the S24 determinant. In one of these rabbits, the response was boosted with a covalent conjugate of S82 and methylated BSA (MBSA). We also measured antibodies to the S24 determinant in rabbit antisera to human, monkey, dog, bovine, and the large and small forms of rat MBP. By nonequilibrium inhibition analysis, we determined that the antibody responses to these antigens were all directed to a determinant composed of residues 66-71 of MBP, and that intact MBP inhibits the binding of these antibodies to radiolabeled S24. The results demonstrate that the rabbit encephalitogenic region of myelin basic protein is exposed in the intact molecule both as an immunogen and as a reactant in liquid-phase assays; furthermore, they demonstrate that MBP antigenicity leading to B cell responses does not necessarily depend on sequence differences between the injected protein and its counterpart in the host species. The latter finding reinforces the contention of Atassi that autoantibody responses are not exclusive to "evolutionary hypervariable locations."     

Immunosorbent method for the detection of A, B, O blood group specificity on CEA preparations.

The detection of A,B,O blood group specificity on some CEA preparations was carried out with anti-A,B,H antisera coupled to sepharose 4B and (¹²⁵I)-labelled antigens. This method was compared to classical FARR's method using ammonium sulfate precipitation of the antigen-antibody complex. In this last procedure, the introduction of adjuvants (excess of γ-globulin or saliva perchloric extract) limits its sensitivity. The use of immunosorbents which allows to eliminate these adjuvants, makes the results more reproducible and the sensitivity higher. Using this method, A blood group determinant was identified in two CEA preparations. Moreover, binding inhibition of labelled A blood group substance to anti-A antiserum by these CEA corroborated this result.     

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49

Three antisera to myelin basic protein—a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP—were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69–84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69–71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69–71. S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.This work supported at Duke University Medical Center by Research Grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service and the Medical Scientist Training Program Grant #5-T32-OMO-7171-08; at St. Luke's Hospital Center by NS-15322 from the National Institutes of Health of the U.S. Public Health Service; and at Northwestern University by Research Grant NS-06262 from the National Institutes of Health of the U.S. Public Health Service.     

Lipid-induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein

The precipitation by antibodies to intact myelin basic protein (BP) and to synthetic peptides containing a sequence based on the region 65 to 83 of bovine BP, S82, S81, S79, and S24, of intact BP in solution or bound to lipid vesicles was compared, using 125I-BP or 14C-DPPC-labeled lipid-BP vesicles. The antipeptide antibodies were shown earlier to recognize conformational determinants which are not expressed in the intact protein in solution. Several anti-BP antibodies precipitated more of the BP free in solution than when bound to lipid vesicles, suggesting that some of the determinants recognized by these antibodies were either sequestered in the bilayer or were altered in conformation. In contrast, one anti-peptide antisera, which had a high titer for the conformational determinant in two of these peptides, S82 and S81, precipitated the protein to a significant degree when it was bound to PG vesicles, even though it did not react with the intact protein in solution. These results indicated that PG was able to confer on the protein the unique peptide conformation recognized by this antibody. PS was less effective, and other lipids were ineffective at conferring this conformation on the protein, supporting earlier results which showed that the conformation of the protein is influenced by the lipid composition of its environment. None of the other anti-peptide antibodies studied bound to the protein either in solution or in lipid vesicles. These results indicate that the lipid environment can sequester or alter the conformation of some antigenic determinants, preventing recognition by some anti-BP antibodies, and can expose or generate other conformational determinants, allowing recognition by an anti-peptide antiserum.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Bombesin receptor from Swiss 3T3 cells. Affinity chromatography and reconstitution into phospholipid vesicles

Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys3]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [35S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and the eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed [125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity.     

Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: Comparison with liver and kidney membrane subunits by two-dimensional electrophoresis

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (³H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (¹²⁵I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated M_r? and pI. Of the 33 ³H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with ¹²⁵I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, M_r?, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.     

Diphtheria toxin inhibits the synthesis of myelin proteolipid and basic proteins by peripheral nerve in vitro

Abstract— Diphtheria toxin (DT) did not produce measurable degradation of myelin proteins or sulphatide in sciatic nerves of chick embryos after incubation in vitro for 4 h. In contrast, DT inhibited the in vitro incorporation of L-[U-¹⁴C]leucine into myelin proteins by the nerves after a delay of 1 h. Separation of the myelin proteins by SDS-polyacrylamide gel electrophoresis indicated that the synthesis of Wolfgram proteins and proteins not entering the gel was inhibited by 21–22 per cent, whereas synthesis of myelin proteolipid and basic proteins was inhibited by 79–88 per cent. Incorporation of ³⁵SO₄ into myelin [³⁵S]sulphatide was also inhibited by DT after a delay of 2 h. The inhibition of [³⁵S]sulpha-tide incorporation into myelin caused by DT differed from that observed with puromycin in that it did not depend on depletion of an intracellular transport lipoprotein. Instead, the inhibition seemed to be secondary to the decreased synthesis of myelin proteolipid and basic proteins.     

Transport of sulphate, thiosulphate and iodide by choroid plexus in vitro

—Isolated choroid plexuses of rabbits and cats were incubated in artificial cerebrospinal fluid medium containing [³⁵S]sulphate, [³⁵S]thiosulphate or [¹²⁵I]iodide and combinations thereof. After 1 hr incubation the mean ratio of tissue concentration to medium concentration was 2·46 for [³⁵S]sulphate, 2·39 for [³⁵S]thiosulphate, and 270 for [¹²⁵I]iodide. Uptake of all three anions was greatly reduced at 0° and by addition of dinitrophenol to the medium. Other inhibitors selectively reduced the uptake of particular anions; non-radioactive sulphate and thiosulphate reduced both [³⁵S]sulphate and [³⁵S]-thiosulphate uptake with much less effect on [¹²⁵I]iodide uptake, while non-radioactive iodide and thiocyanate greatly reduced [¹²⁵]iodide uptake with little or no effect on [³⁵S]sulphate or [³⁵S]thiosulphate uptake. It was concluded: (a) that sulphate and thiosulphate, like iodide, were accumulated by choroid plexus in vitro by active transport; (b) that sulphate and thiosulphate share and compete for a transport mechanism which is separate from the iodide transport mechanism; and (c) that the transport of sulphate out of cerebrospinal fluid demonstrated in vivo could occur at least in part in the choroid plexus.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity in Chinese hamster ribosomal DNA

A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [¹²⁵I]18S or [¹²⁵I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5×10⁶ daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.     

Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/- mice

A neutralizing antibody to the receptor for the advanced glycation end products (anti-RAGE Ab) was developed as a potential treatment of acute and chronic inflammatory conditions. Previous pharmacology studies demonstrated efficacy of the anti-RAGE antibody in the mouse model of sepsis. We examined pharmacokinetics and lung distribution of [¹²⁵I]anti-RAGE Ab in RAGE-/- and wild-type (129S5) mice following single IV administration. Serum pharmacokinetics of [¹²⁵I]anti-RAGE Ab was similar in RAGE-/- and 129S5 mice, with the total body clearance of 0.3 mL/hr/kg and the elimination half-life of 11-12 days, suggesting the target expression had limited impact on overall elimination of [¹²⁵I]anti-RAGE Ab from mice. [¹²⁵I]Anti-RAGE Ab accumulated in the lung of 129S5 mice, with ~4% of total dose retained in the lung at days 6-27 and the lung AUC0-∞ of ~300% of that in serum. The SDS-PAGE analysis suggested that most of retained lung radioactivity was attributed to intact antibody. No accumulation of radioactivity was observed in the lung of RAGE-/- mice, indicating that lung uptake of [¹²⁵I]anti-RAGE Ab was target-dependent in wild-type mice. These data suggest that the anti-RAGE Ab was able to localize to the site of RAGE expression, the lung, and support the findings in the previous pharmacology studies.     

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

Abstract

We have investigated the binding characteristics of rat [¹²⁵I] adrenomedullin (AM) and human [¹²⁵I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [¹²⁵I] rat AM and [¹²⁵I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [¹²⁵I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [¹²⁵I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [¹²⁵I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [¹²⁵I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Antibodies to nicotinic acetylcholine receptors used to probe the structural and functional relationships between brain α-bungarotoxin binding sites and nicotinic receptors

Antibodies against peripheral nicotinic acetylcholine receptors (nAChR) were used to determine the proportion of brain α-bungarotoxin binding sites that are immunologically related to the peripheral nAChR. The α-bungarotoxin binding component partially purified from rat brain was labelled with [¹²⁵I]α-bungarotoxin and reacted with increasing concentrations of rabbit anti(nAChR) antisera. At least 75% of the brain protein could be immunoprecipitated by rabbit anti(rat muscle junctional nAChR) antiserum (M) whereas an antiserum against Torpedo nAChR (J) was without effect and clearly failed to cross-react with the brain component. Both antisera precipitated 100% of [¹²⁵I]α-bungarotoxin-labelled nAChR from Torpedo marmorata. The lower precipitation of the brain protein was not a consequence of [¹²⁵I]α-bungarotoxin dissociating during the precipitation. We conclude that the majority of α-bungarotoxin binding sites in brain are clearly recognised by the crossreacting antiserum.Release of [³H]dopamine from striatal synaptosomes could be elicited by nicotine in a dose-dependent manner and the response was prevented by the ganglionic blocker mecamylamine, although antagonism by α-bungarotoxin was less clearcut. Preincubation of the synaptosomes with antiserum M resulted in a statistically significant decrease in the [³H]dopamine response to nicotine at all agonist concentrations tested. Antiserum J, however, had no consistent effect on the response. Thus the actions of the antisera parallel their ability to recognise the brain α-bungarotoxin binding component. We conclude that the cholinergic regulation of dopamine release is in part mediated through a nAChR that is immunologically related to the nAChR of the neuromuscular junction and to the α-bungarotoxin binding component that can be isolated from rat brain.