N-succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates: a new class of reagents for protein radioiodination

N-Succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates (alkyl = Me, Bu) have been prepared and used as a precursor to label N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). SIPC was obtained in greater than 80% yield from either the methyl or butyl precursor with N-chlorosuccinimide and heating at 60-65 degrees C. Significantly lower yields were observed with tert-butyl hydroperoxide. After a 30-min incubation with [131I]SIPC at pH 8.5, goat IgG, an intact monoclonal antibody (MAb), and a MAb F(ab')2 fragment were labeled in 60-65% yield. Specific binding of the MAb and MAb fragment after SIPC labeling was identical with that observed with N-succinimidyl 3-iodobenzoate and higher than that reported previously for these MAbs after labeling by using the Iodogen method. When 5-[131I]iodonicotinic acid was injected into normal mice, thyroid uptake was less than 0.2% of the injected dose, reflecting the inertness of this compound to deiodination. Paired-label biodistribution studies indicate that for both the MAb and the F(ab')2 labeled by using SIPC, accumulation of activity in the thyroid and other tissues is comparable to that observed when these proteins were labeled by using N-succinimidyl 3-iodobenzoate. The results of this study suggest that SIPC may be a reagent for labeling MAbs with halogen nuclides.     

In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain

In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.     

Interaction of benzanthrone with cytochrome P450: altered patterns of hepatic xenobiotic metabolism in rats

Benzanthrone, an anthraquinone dye intermediate, is commonly used for the synthesis of a number of polycyclic vat and disperse dyes. Our prior studies have shown that benzanthrone can be metabolized by rat hepatic microsomal cytochrome P450 (P450) (Biochem. Int., 18, 1989, 1237). In this study, the interaction of benzanthrone with rat hepatic microsomal P-450 and its effect on xenobiotic metabolism have been investigated. Parenteral administration of benzanthrone (40 mg/kg body weight) for 3, 7, or 21 days caused no change in the relative body weight or organ weight of rats. The levels of P450 were found to be reduced (33%-50%) in all the benzanthrone-exposed animals at all the time periods. In vitro addition of benzanthrone caused a spectral change with oxidized P450 and concentration-dependent reduction in the carbon monoxide spectrum of dithionite-reduced P450. The addition of benzanthrone to hepatic microsomes prepared from phenobarbital-treated rats resulted in spectral changes characterized by an absorbance maximum at 397 nm indicative of type I binding. In vitro addition of benzanthrone showed a concentration-dependent inhibition of hepatic aminopyrine N-demethylase (APD) and ethoxyresorufin-O-deethylase (ERD) activities with respective I50 values of 9.5 x 10(-4) and 8.0 x 10(-5) M. However, the inhibition of aryl hydrocarbon hydroxylase (AHH) even at the highest concentration of benzanthrone (10(-2) M), was of the order of only 29%. In vivo administration of benzanthrone also led to the inhibition of APD, AHH, and ERD activities at all treatment times although the magnitude of inhibition was of a lower order.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potato Virus X—a New Report from Turnip (Brassica rapa L.) in India

A virus isolated from turnip in Aligarh, India, which caused mild mosaic, mottling and curling of leaves followed by overall stunting of plants, was characterized as potato virus X (PVX) on the basis of its host range, biological and physical properties, particle morphology, ultrastructural studies, and serological relationship.     

Flexibility in the donor substrate specificity of {beta} 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates

Biosynthetically, bovine N-acetylglucosainine ß 1,4-galacto-syltransferase(GalT) catalyses the transfer of galactosyl residues from UDP-Galto the 4-position of GlcNAc units, resulting in the productionof N-acetyllactosamine sequences. UDP-Glc and UDP-GalNAc werealso found to act as donors for this enzyme, allowing the preparationof ßGlc(14)-ßGlcNAc and ßGalNAc(14)ßGlcNActerminating structures on the milligram scale. GalT could thusbe used to add ßGalNAc to ßGlcNAc(12)Manterminating structures, converting them to the ßGalNAc(14)ßGlcNAc(12)Mansequences found on glycoprotein hormones. GalT did not transferGlcNAc residues from UDP-GlcNAc, but it could utilize UDP-GlcNH₂as a donor. Synthesis of ßGlcNAc(14)ßGlcNAcsequences could therefore be accomplished by transfer of GlcNH₂from its UDP derivative, followed by N-acetylation of the productamino-disaccharide using acetic anhydride in methanol. The productsof the enzymatic reactions were characterized by ¹H-NMR-spectroscopyand fast-atom bombardment mass spectrometry. This work expandsthe scope of the combined chemical-enzymatic synthesis of complexcarbohydrates, using glycosyltrans-ferases, to the productionof oligosaccharides different from those for which these enzymeswere designed. These unnatural reactions should find applicationin glycoprotein and glycolipid remodelling. galactosyltransferase chemica1-enzymatic synthesis of oligosaccharides oligosaccharide analogues sugar-nucleotide analogues carbohydrate remodelling     

Nitric oxide-generating vasodilators inhibit mitogenesis and proliferation of BALB/C 3T3 fibroblasts by a cyclic GMP-independent mechanism.

The purpose of this study was to investigate the effects of nitric oxide-generating vasodilators and 8-bromo-cGMP on serum-induced mitogenesis in BALB/c 3T3 fibroblasts that lack soluble guanylate cyclase activity. Two such vasodilators, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate, decreased the incorporation of (3H)thymidine in these cells dose-dependently whereas 8-bromo-cGMP was ineffective at concentrations of up to 10 mM. Moreover, S-nitroso-N-acetylpenicillamine also inhibited cell proliferation, consistent with the data on (3H)thymidine incorporation. S-nitroso-N-acetylpenicillamine had no effect on cGMP accumulation, confirming previous studies that these cells lack soluble guanylate cyclase activity. Hemoglobin and FeSO4/ascorbate, agents that inhibit the actions of nitric oxide, both decreased S-nitroso-N-acetylpenicillamine-induced antimitogenesis, supporting the view that this effect was related to the generation of nitric oxide. The antimitogenic activity of S-nitroso-N-acetylpenicillamine was unlikely to be the expression of nitric oxide-induced degradation of serum mitogens, as indicated by the decrease of the antimitogenic activity on prolonged preincubation of SNAP in serum-containing medium. We conclude that nitric oxide-generating vasodilators inhibit serum-induced mitogenesis and cell proliferation in BALB/c 3T3 fibroblasts by a cGMP-independent mechanism.     

Effect of cultural factors on cellulase activity and protein production by Aspergillus terreus

Protein production by Aspergillus terreus GN1 grown on 1.0% alkali-treated bagasse was studied under various cultural conditions. The maximum biomass protein content of 20.1% and protein recovery of 11.2% was obtained with an initial pH of 4.0, with 1/5 (v/v) inoculum in continuously shaken cultures grown for seven days. Protein content of the alkali-treated bagasse was 3.0%. Highest crude protein percent also corresponded with highest carboxymethyl cellulase and filter paper enzyme activities.     

E. coli membranes become permeable to ions following T7-virus-infection

Summary Infection of E. coli with the viruses T7 or T3 leads to a dramatic efflux of potassium ions. This ion efflux is caused by the virus particle since no concomitant protein synthesis is required. T7 mutants carrying deletions in the M-gene (Schweiger et al., 1975), however, yield virus particles disturbed in the ion release.