Immunological studies on nucleic acids and their components.8.Antibodies specific to a deoxyribotrinucleotide sequence

Bovine serum albumin conjugates of two trinucleotides, dpTpTpA and dTpTpAp, were prepared by linking the trinucleotides through their end phosphates by the ‘carbodiimide method’. Antibodies were raised in rabbits by injecting the trinucleotide-bovine serum albumin conjugates. Analysis by double diffusion in agar gel, quantitative precipitin reaction and its inhibition by haptens showed clearly the presence of antibodies specific to the whole trinucleotide molecule. The titre of antibodies obtained by the trinucleotide-rabbit serum albumin conjugates with their respective antisera was approximately the same, indicating that linking the trinucleotide through either 5′ or 3′ phosphate does not have an appreciable effect on the titre of antibodies. The results also demonstrate that the nucleotide(s) away from the carrier protein is more immunodominant than the one linked directly to the protein.

Immunologic studies on nucleic acids and their components. I. An analysis of the specificity of anti-deoxyadenylate antibodies by a membrane-binding technique

Anti-deoxyadenylate antibodies were produced in rabbits by injecting a conjugate of deoxyadenosine 5′-phosphate with bovine serum albumin. The antisera, as analyzed by double diffusion in agar and the quantitative precipitin reaction, showed hapten-specific antibodies. The specific interaction between [3H]deoxyadenylate and antiserum was studied by a sensitive nitrocellulose membrane-binding assay. The specificity of the antibodies was analyzed by measuring the effectiveness of other nucleotides or derivatives to inhibit the hapten-antibody binding. The requirements for recognition by the antibody sites were studied by using a series of naturally occurring nucleic acid components as well as some synthetic derivatives as inhibitors. The antibodies were found to show a high degree of specificity for the whole nucleotide, the base, sugar and phosphate playing almost equally important roles. There was cross reactivity with other mononucleotides, although of a low order. The antibodies were able to react with DNA and tRNA.

Immunologic studies on nucleic acids and their components II. Reversible inhibition of anti-nucleoside antibodies in aqueous pyridine and its application in antibody

The effect of aqueous pyridine on a hapten—antihapten system was investigated by the quantitative precipitin reaction and by the membrane filtration method. It was found that dilute solutions of pyridine inhibited the reaction between isopentenyladenosine and its antiserum. Other solvents examined were less effective. The effect of pyridine was reversible at concentrations where complete inhibition occurred, thus indicating its use for the dissociation of antigen—antibody complexes. The inhibitory effect of pyridine was exploited in a single-step purification method for anti—isopentenyladenosine and antideoxy-adenylate antibodies. In addition, generally applicable methods for linking nucleosides and nucleotides to aminoethyl-Sepharose are described.

Immunologic studies on nucleic acids and their components II. Reversible inhibition of anti-nucleoside antibodies in aqueous pyridine and its application in antibody purification

The effect of aqueous pyridine on a hapten—antihapten system was investigated by the quantitative precipitin reaction and by the membrane filtration method. It was found that dilute solutions of pyridine inhibited the reaction between isopentenyladenosine and its antiserum. Other solvents examined were less effective. The effect of pyridine was reversible at concentrations where complete inhibition occurred, thus indicating its use for the dissociation of antigen—antibody complexes. The inhibitory effect of pyridine was exploited in a single-step purification method for anti—isopentenyladenosine and antideoxy-adenylate antibodies. In addition, generally applicable methods for linking nucleosides and nucleotides to aminoethyl-Sepharose are described.

On the Specificity of Carbohydrate-Lectin Recognition The Interaction of a Lectin from Ricinus communis Beans with Simple Saccharides and Concanavalin A

A galactose-specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature-dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively. The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A-specific sugars. The complex formation results in a time-dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time-dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein-protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell-lectin interaction is discussed.

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli. Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli, respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.

A New Tetrahydrofurannulation Via Tandem Radical Cyclization Reaction-Reductive Deoxygenation Sequence

Treatment of bromoketals 2, derived from allyl alcohols 1, with tributyltin chloride, sodium cyanoborohydride and AIBN furnishes the tetrahydrofurannulated products 3 via a 5-exo-trig radical cyclisation reaction followed by reductive cleavage of ketal 4.

Vanadium Catalysis in Bromoperoxidation Reaction

Peroxidative bromination of phenol red to its tetrabromo derivative, bromophenol blue, required vanadate in addition to H2O2 when carried out in the pH range of 5-7. Excess H2O2, with ratio of H2O2:vanadate of 2:1 and above, prevented the reaction. Diperoxovanadate, known to be formed in such reaction mixtures, was ineffective by itself and needed uncomplexed vanadate (V-v) or vanadyl (V-iv) to support bromination. Bromide-assisted reduction of the excess vanadate to vanadyl appeared to be an essential secondary reaction. In the absence of phenol red oxygen was released, and concomitantly bromide was oxidized to a form competent to brominate phenol red added after termination of oxygen release. These findings indicated participation of reactions leading to an intermediate derived from vanadyl and diperoxovanadate, previously described from this laboratory (Arch. Biochem. Biophys. 316, 319-326, 1995). Continuous bromination of phenol red occurred when glucose oxidase-glucose system was used as a source of continuous flow of H2O2. A scheme of reactions involving peroxovanadates (mono-, di-, mu-, and bromo-) is proposed for the formation and utilization of an active brominating species and for the recycling of the product, mono-peroxovanadate, by H2O2, which explains the catalytic role of vanadium in the bromoperoxidation reaction.

Nanoporous Pt with High Surface Area by Reaction-Limited Aggregation of Nanoparticles

Nanoporous structures with high active surface areas are critical for a variety of applications. Here, we present a general templateless strategy to produce such porous structures by controlled aggregation of nanostructured subunits and apply the principles for synthesizing nanoporous Pt for electrocatalytic oxidation of methanol. The nature of the aggregate produced is controlled by tuning the electrostatic interaction between surfactant-free nanoparticles in the solution phase. When the repulsive force between the particles is very large, the particles are stabilized in the solution while instantaneous aggregation leading to fractal-like structures results when the repulsive force is very low. Controlling the repulsive interaction to an optimum, intermediate value results in the formation of compact structures with very large surface areas. In the case of Pt, nanoporous clusters with an extremely high specific surface area (39 m(2)/g) and high activity for methanol oxidation have been produced. Preliminary investigations indicate that the method is general and can be easily extended to produce nanoporous structures of many inorganic materials.

Enantiospecific approach to the tricyclic core structure of tricycloillicinone, ialibinones, and takaneones via ring-closing metathesis reaction

Enantiospecific synthesis of the tricyclic core structure present in the biologically active natural products tricycloillicinone, ialibinones, and takaneones, starting from the readily available campholenaldehyde employing a transannular RCM reaction as the key step, has been accomplished.

Acid-catalysed cyclisations: structures of novel products isolated in the reaction of 2-[3-(2,6-dimethoxyphenyl)but-2-enyl]-2-methylcyclopentane-1,3-dione with MeOH-HCl

Reaction of the title compound (1a) with anhydrous MeOH-HCl gave 2-endo-(2,6-dimethoxyphenyl)-2-exo-methyl-5-methylbicyclo[3.2.1]octane-6,8-dione (3a), 1,5,14-timethoxy-5,8-seco-6,7-dinorestra-1,3,5(10),9(11)-tetraen-17-one (4), 1,5-dimethoxy-5,8-seco-6,7-dinorestra-1,3,5(10),8,14-pentaen-17-one (5), and 3,4,5,6-tetrahydro-2,7-dimethoxy-3,6-dimethyl-3,2,6-(13-oxopropan[1]yI[3]ylidene)-2H-1-benzoxocin (6). Structures assigned to compounds (3a), (4), and (6) are based on spectral data. The exo-tricyclic acetal structure (6) was further confirmed by the analysis of the 1H n.m.r. spectra of the isomeric alcohols (11) and (12), obtained by sodium borohydride reduction of (6).

Vilsmeier reaction on some 6- and 7-methoxy-1- and 2-tetralones.

Vilsmeier reaction on a few representative 6- and 7-methoxy-1- and 2-tetralones has been investigated. While 1-tetralones give the corresponding 1-chloro-2-formyl3, 4-dihydronaphthalenes, the 2-tetralones afford 1,3-bisformyl-2-chloronaphthalenes. Spectral characteristics of all the products obtained are given and a mechanistic proposal has been made to explain the observed chlorobisformylation.