Nifurtimox nitroreductase activity in different cellular fractions from male rat pancreas. Biochemical and ultrastructural alterations

Nifurtimox (Nfx) is a nitroheterocyclic drug used in the treatment of Chagas' disease. It has serious side effects which frequently force to interrupt the treatment. Nfx toxicity has been linked to its nitroreduction to a nitroanion radical with a subsequent redox cycling which generate reactive oxygen species. We analyzed the ability of Sprague Dawley male rat pancreas to nitroreduce Nfx and whether this drug may cause deleterious effects in this organ. The microsomal fraction exhibited Nfx nitroreductase activity in the presence of NADPH under anaerobic atmosphere, which was fully inhibited under air but not altered when N2 was replaced by pure CO. The cytosol nitroreduced Nfx in the presence of hypoxanthine under N2; it was inhibited by allopurinol and negligible in aerobiosis. Nfx reached pancreatic tissue at 1, 3 or 6 h after intragastric administration (100 mg/kg). Six hours after drug administration, a significant increase in t-buthylhydroperoxide promoted chemiluminiscence was detected. Pancreatic protein sulfhydryl content significantly decreased at either 1, 3 or 6 h after Nfx administration. No changes in either protein carbonyl or in lipid hydroperoxides were observable. Ultrastructural alterations were observed in the endoplasmic reticulum and nuclei from acinar cells and in the insulin-containing granules from the pancreas. However, the seric amylase levels were not changed, but the blood glucose levels were slightly but significantly increased 24 h after Nfx administration. These studies might suggest that Nfx treatment could impose an increased risk to patients exposed to other insults provoking oxidative stress or having preexisting pathologies in the pancreas.     

Benzonidazole levels in blood vary with age in rats

Benznidazole (Bz) exhibits toxic side effects in animal studies and clinical use. Reductive metabolism of Bz in liver microsomes modulates the duration of its chemotherapeutic effect and its toxicity. The rate of this metabolism depends on age and is less intense in newborns and youngsters than in adults. In the present study, we determined Bz blood levels in rats of different ages that received Bz intragastrically (100 mg/kg). We developed and validated a high-pressure liquid chromatography with UV detector method for determination of Bz levels in whole blood. Bz levels were significantly higher and persisted for longer periods of time in the blood of young rats when compared to that of adult animals.     

狼毒活性部位复合乳油功效

为增强瑞香狼毒制剂的杀虫药效、充分开发利用以瑞香狼毒为主的植物源农药,进行了瑞香狼毒杀虫活性部位与其它药物的复配实验。采用瑞香狼毒杀虫活性部位(LD)分别与烟碱乳油(YJR)、CT-901A(Bz)的不同质量比,在室内对家蝇、酢酱草茹叶螨(Petrbia hartiEwing)进行毒力测定,利用软件进行毒力回归分析。实验结果显示,LD和YJR、LD和Bz的两两复配及三者(LD YJR Bz)的复配能够起显著增效作用,特别是20:1:1(LD:YJR:Bz)、30:1:1(LD:YJR:Bz)对家蝇的毒力效果更加明显,LC50达到了0.005 mg/L,20:1(LD:Bz)、30:1(LD:Bz)、30:1:1(LD:YJR:Bz)对酢酱草茹叶螨(Petrbia hartiEwing)的毒力效果也同样显著,LC50也达到了0.005 mg/L。并对瑞香狼毒活性部位进行初步分离检测,其有效成分为黄酮类物质。     

Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones

The gene nfxB is one of the genes which affect the cell membrane permeability of quinolones in Pseudomonas aeruginosa PAO. Both wild-type nfxB and a mutant nfxB (nfx13E) were cloned and the DNA sequences were determined. The wild-type gene was dominant in PAO strains. The nfxB mutation was a point mutation (cytosine----guanine) which generates an amino acid exchange (arginine----glycine) in the putative nfxB product. The amino acid sequence of the wild-type NfxB protein revealed that it has a helix-turn-helix motif which may be responsible for the ability to bind in a sequence-specific manner to DNA. This finding indicated that the NfxB protein may regulate the expression of genes that are associated with cell permeability of drugs in P. aeruginosa. The position of the amino acid substitution between the NfxB protein and the Nfx13E protein was located within a possible DNA-binding domain, suggesting that the mutant protein (Nfx13E) may have lost DNA binding ability and regulator activity.     

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.     

Photochemical probes of the active site of myosin. Irradiation of trapped 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate labels the 50-kilodalton heavy chain tryptic peptide

3'-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP), an analog of ATP containing a photoreactive benzophenone moiety, was used as a probe of the ATP binding site of myosin subfragment 1 (SF1). The inactivation of SF1 NH+4-EDTA ATPase by the bifunctional thiol crosslinking system cobalt(II)/cobalt(III) phenanthroline complexes was enhanced by Bz2ATP to the same degree as by ATP. This treatment resulted in the stable trapping of Bz2ATP at the active site in nearly stoichiometric amounts in a manner exactly analogous to ATP (Wells, J.A., and Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970). Irradiation of SF1 containing trapped [3H]Bz2ATP gave approximately 50% covalent incorporation of the trapped nucleotide into the enzyme. Analysis of photolabeled SF1 by gel electrophoresis showed that all of the [3H]Bz2ATP was attached to the 95-kDa heavy chain fragment. No label was found in the light chains. Similar analysis of the same protein after limited trypsin treatment demonstrated that approximately 75% of the [3H]Bz2ATP was bound to the central 50-kDa peptide and its 75-kDa precursor from the heavy chain. The N-terminal 25-kDa tryptic peptide, shown to be photolabeled by other ATP analogs (Szilagyi, L., Balint, M., Sreter, F.A., and Gergely, J. (1979) Biochem. Biophys. Res. Commun. 87, 936-945; Okamoto, Y., and Yount, R.G. (1983) Biophys. J. 41, 298a), was not labeled (less than 1%) by Bz2ATP. These results demonstrate that portions of the 50 kDa-peptide of the heavy chain are within 6-7 A of the ATP binding site on SF1 and possibly contribute to nucleotide binding.     

Three-centre, two-electron bonds in complexes of Mn, Ni, Co and Cd with 3-(4-benzonitrile)pyrazolyl scorpionates

A series of complexes of the form [M(Bp^3(4Bz))(Tp^3(4Bz))] containing a mixture of novel dihydrobis(3-(4-benzonitrile)pyrazolyl)borato (Bp^3(4Bz)) and hydrotris(3-(4-benzonitrile)pyrazolyl)borato (Tp^3(4Bz)) ligands have been synthesised and structurally characterised. The ligands, both containing 3-(4-benzonitrile)pyrazole arms, form isostructural complexes with a variety of transition metals although different crystalline solvates have been obtained depending upon the crystallisation conditions, namely [M(Bp^3(4Bz))(Tp^3(4Bz))] (M = Ni, Co), [Ni((Bp^3(4Bz))(Tp^3(4Bz))]·2MeOH and [M(Bp^3(4Bz))(Tp^3(4Bz))]·MeOH (M = Mn, Cd). In all cases the metal atoms are five-coordinate with an additional agostic B-H interaction from the bis(pyrazolyl)borate ligand.     

Probing the structure of the nicotinic acetylcholine receptor with 4-benzoylbenzoylcholine, a novel photoaffinity competitive antagonist

[(3)H]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K(D) = 1.4 microm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [(3)H]Bz(2)choline results in the covalent incorporation of [(3)H]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [(3)H]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gammaVal-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at deltaAsp-99. Amino-terminal sequence analysis identified the homologous residues gammaLeu-109 and deltaLeu-111 as the primary sites of [(3)H]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gammaLeu-109/deltaLeu-111.     

Ruthenium Complex with Benznidazole and Nitric Oxide as a New Candidate for the Treatment of Chagas Disease

Background

Chagas disease remains a serious medical and social problem in Latin America and is an emerging concern in nonendemic countries as a result of population movement, transfusion of infected blood or organs and congenital transmission. The current treatment of infected patients is unsatisfactory due to strain-specific drug resistance and the side effects of the current medications. For this reason, the discovery of safer and more effective chemotherapy is mandatory for the successful treatment and future eradication of Chagas disease.

Methods and Findings

We investigated the effect of a ruthenium complex with benznidazole and nitric oxide (RuBzNO₂) against Trypanosoma cruzi both in vitro and in vivo. Our results demonstrated that RuBzNO₂ was more effective than the same concentrations of benznidazole (Bz) in eliminating both the extracellular trypomastigote and the intracellular amastigote forms of the parasite, with no cytotoxic effect in mouse cells. In vivo treatment with the compound improved the survival of infected mice, inhibiting heart damage more efficiently than Bz alone. Accordingly, tissue inflammation and parasitism was significantly diminished after treatment with RuBzNO₂ in a more effective manner than that with the same concentrations of Bz.

Conclusions

The complexation of Bz with ruthenium and nitric oxide (RuBzNO₂) increases its effectiveness against T. cruzi and enables treatment with lower concentrations of the compound, which may reduce the side effects of Bz. Our findings provide a new potential candidate for the treatment of Chagas disease.     

Photolabeling of the 6 and 10 S conformations of gizzard myosin with 3'(2')-O-(4-Benzoyl)benzoyl-ATP. Proline 324 is near the active site

3'(2')-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP) was used as a photoaffinity label of the ATP binding site of unphosphorylated chicken gizzard myosin. Specific photolabeling of the active site of 6 S myosin was assured by forming a stable myosin.Co(II)Bz2ADP.orthovanadate complex (termed trapping) prior to irradiation. Co2+ was used in place of Mg2+ to prevent the known photoreaction of vanadate with myosin which destabilizes the trapped complex. [3H] Bz2ADP.Pi was also stably trapped on gizzard myosin by forming the 10 S folded conformation of the protein in the presence of [3H]Bz2ATP and Mg2+. Irradiation of 6 S myosin containing orthovanadate trapped [3H] Bz2ADP or 10 S trapped [3H]Bz2ADP.Pi gave 32 and 30% covalent incorporation, respectively. The 50-kDa and precursor 68-kDa tryptic peptides of the subfragment-1 heavy chain derived from both forms of myosin were found to contain essentially all of the covalently attached [3H]Bz2ADP. Parallel experiments with untrapped [3H]Bz2ADP showed extensive nonspecific labeling of all of the major tryptic peptides and the light chains. Eight labeled peptides, isolated from 6 and 10 S photolabeled myosin, contained the sequence G319-H-V-P-I-X-A-Q326, where X corresponds to labeled proline 324. [14C]Bz2ADP was previously shown to label serine 324 in skeletal subfragment-1 (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995), which corresponds to alanine 325 in the gizzard sequence. Thus, this region of the 50-kDa tryptic fragment, near the nucleotide binding site, in both skeletal and smooth muscle myosins, must fold in essentially the same manner.     

2-Benzoylpyridine-N(4)-tolyl thiosemicarbazones and their palladium(II) complexes: Cytotoxicity against leukemia cells

The palladium(II) complexes [Pd(2Bz4oT)Cl], [Pd(2Bz4mT)Cl], and [Pd(2Bz4pT)Cl] were prepared with N(4)-ortho- (H2Bz4oT) N(4)-meta- (H2Bz4mT) and N(4)-para- (H2Bz4pT) tolyl-thiosemicarbazones derived from 2-benzoylpyridine. The free thiosemicarbazones proved to be highly cytotoxic against Jurkat, HL60 and the resistant HL60.Bcl-X_L leukemia cell lines at nanomolar concentrations, but were much less cytotoxic to HepG2 human hepatoma cells. Upon coordination to palladium(II) the cytotoxic activity against all studied cell lines decreases. However, the high cytotoxicity of the free thiosemicarbazones against leukemia, together with their hepatotoxic profile similar to that of cisplatin suggest that N(4)-tolyl thiosemicarbazones have potential as chemotherapeutic drug candidates.     

The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate

The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.     

31P NMR studies on the putative chemoselective activation of nucleoside-3'-thiophosphate-O-esters

P-diastereomerically pure O-esters of N(Bz)-5'-DMT-dA-3'-monothiophosphate, having charged S=P-O(-) moiety, have been synthesized. Chemoselectivity of their activations by formation of different mixed anhydrides, followed by couplings with N(Bz)-3'-levulinyl-dA, were studied by 31P NMR spectroscopy.     

Chromosome Breakage Undetectable in Active Mu Lines of Maize

We have used a set of Mutator-induced mutants of Bz1 to test whether members of the Mutator (Mu) family of maize transposable elements produce broken chromosomes. From our inability to demonstrate the simultaneous loss of two dominant endosperm markers distal to Mu insertions at Bz1 we conclude that either Mu, unlike many elements of the Ds family, does not induce such breaks, or it does so at a very low frequency.     

Fast deprotection of synthetic oligodeoxyribonucleotides using standard reagents under microwave irradiation

Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz, dC Bz, dC Ac, dG ibu, dG PAC) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz, dC Bz, dG ibu) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170 degrees C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.