An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.     

Euclein: A new binaphthaquinone from Euclea pseudebenus

7-MethyljugIone, 8,8′-dihydroxy-4,4′-dimethoxy-6,6′-dimethyl-2,2′-binaphthyl-1,1′-quinone, 2-methylnaphthazarin, mamegakinone and euclein have been isolated from Euclea pseudebenus. Euclein is the 3,6′-dimer of 7-methyljuglone.     

Towards the mechanism and comparative effect of diphenyl diselenide, diphenyl ditelluride and ebselen under various pathophysiological conditions in rat's kidney preparation

As an extension of our previous work we not only evaluated the relationship between acidosis and lipid peroxidation in rat's kidney homogenate, but also determined for the first time the potential anti-oxidant activity of diphenyl diselenide, diphenyl ditelluride and ebselen at a range of pH values (7.4–5.4). Because of the pH dependency of iron redox cycling, pH and iron need to be well controlled and for the reason we tested a number of pH values (from 7.4 to 5.4) to get a closer idea about the role of iron under various pathological conditions. Acidosis increased rate of lipid peroxidation in the absence Fe (II) in kidney homogenates especially at pH 5.4. This higher extent of lipid peroxidation can be explained by; the mobilized iron which may come from reserves where it is weakly bound. Addition of iron (Fe) chelator desferoxamine (DFO) to reaction medium completely inhibited the peroxidation processes at all studied pH values including acidic values (5.8–5.4). In the presence of Fe (II) acidosis also enhanced detrimental effect of Fe (II) especially at pH (6.4–5.4). Diphenyl diselenide significantly protected lipid peroxidation at all studied pH values, while ebselen offered only a small statistically non-significant protection. The highest anti-oxidant potency was observed for diphenyl ditelluride. These differences in potencies were explained by the mode of action of these compounds using their catalytic anti-oxidant cycles. However, changing the pH of the reaction medium did not alter the anti-oxidant activity of the tested compounds. This study provides evidence for acidosis catalyzed oxidative stress in kidney homogenate and for the first time anti-oxidant potential of diphenyl diselenide and diphenyl ditelluride not only at physiological pH but also at a range of acidic values.     

Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.     

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme

The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which probably plays a role in maintaining the conformation of the kinase sugar phosphate-binding site.     

Oxidative metabolism of spironolactone: evidence for the involvement of electrophilic thiosteroid species in drug-mediated destruction of rat hepatic cytochrome P450

In a preliminary paper [Decker et al. (1986) Biochem. Biophys. Res. Commun. 136, 1162] we have shown that the antimineralocorticoid spironolactone (SPL) preferentially inactivates dexamethasone (DEX) inducible rat hepatic cytochrome P450p isozymes in a suicidal manner. These findings are now confirmed, and the kinetic characteristics of such a process are detailed. In an effort to elucidate the mechanism of SPL-mediated inactivation of cytochrome P450, we have examined the metabolism of SPL in vitro. Incubation of [14C]SPL and NADPH with liver microsomes prepared from DEX-pretreated rats results in the formation of several polar metabolites separable by HPLC with UV detection. This process is found to be dependent on NADPH, O2, SPL, and enzyme concentration, as well as temperature. Furthermore, metabolite formation was significantly attenuated by P450 inhibitors CO and n-octylamine. Mass spectral analysis (thermospray LC/MS, FAB/MS, and FAB/MS/MS) of the two most prominent polar metabolites indicated that these compounds had molecular weights that corresponded to the sulfinic and sulfonic acid derivatives of deacetyl-SPL (SPL-SH). These findings document the formation of previously unreported polar metabolites of SPL by rat liver microsomes enriched in cytochrome P450p and implicate a role for this isozyme in the oxidation of the thiol moiety of deacetyl-SPL. The detection of such metabolites also implicates a catalytic trajectory that includes the thiyl radical and/or sulfenic acid species as a plausible protagonist in drug-mediated inactivation of cytochrome P450p.     

Role of gastrin/pentagastrin in regulation of intestinal cytochrome P-450

1. In the absence of intraluminal inducers, low "basal" levels of cytochrome P-450 and its dependent MFO activities are detected in the rat intestinal mucosa, and may be regulated by endogenous hormones. 2. Rats were nutritionally maintained by either short term (48 hr) intravenous glucose infusion or chronic (8 days) intravenous hyperalimentation, and were treated with various doses of pentagastrin in the infusate. 3. Regardless of the dose (6-90 micrograms/kg/hr) or duration of infusion (2-8 days), pentagastrin had no effect on small intestinal cytochrome P-450, its dependent MFO activity, or the activity of delta-aminolevulinic acid synthetase. 4. The intestinal trophic peptide hormone, gastrin, apparently does not regulate the cytochrome P-450-dependent MFO system of the small intestine.     

Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.