Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0 mg/kg. Lungs and livers were harvested after 24 h, the DNA extracted and subjected to ³²P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50 mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.     

Human mycoses in Portugal [1960–1973]

A critical survey is made of human mycoses diagnosed in European Portugal and in the Portuguese Overseas Provinces. Dermatophyte infections and pityriasis versicolor are commom in the entire territory. The more frequently isolated dermatophytes in Continental Portugal wereTrichophyton violaceum, Microsporum canis, Trichophyton tonsurans andTrichophyton schoenleinii, in the scalp andTrichophyton rubrum, Trichophyton mentagrophytes, Trichophyton megninii andEpidermophyton floccosum in the other body sites. In the Overseas Provinces the species found in white people were very much the same, whereas in negroes mainlyMicrosporum audouinii andT. violaceum in Mozambique,M. audouinii in Angola, andTrichophyton soudanense, M. audouinii andT. rubrum in Guinea were identified.In Continental Portugal cases of candidiasis, mycetoma, aspergillosis, sporotrichosis and cryptococcosis were described in patients who had never been outside Europe; and tinea nigra, African histoplasmosis and South-American blastomycosis in individuals who live or lived in India, Africa and Brazil.The first case of North-American blastomycosis was reported in Mozambique.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Proton magnetic resonance studies of 7 Fe ferredoxins: Lower potential of the [4 Fe–4 S] redox center than the [3 Fe–3 S] cluster

Two types of iron-sulfur clusters, [3 Fe–3 S] and [4 Fe–4 S], were identified by ¹H-NMR in ferredoxins from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. The [4 Fe–4 S] clusters always showed the redox couples which had potentials lower than that of the [3 Fe–3 S] clusters.     

Antibacterial activities of Groebke–Blackburn–Bienaymé-derived imidazo[1,2-a]pyridin-3-amines

We sought to explore the imidazo[1,2-a]pyridin-3-amines for TLR7 (or 8)-modulatory activities. This chemotype, readily accessed via the Groebke–Blackburn–Bienaymé multi-component reaction, resulted in compounds that were TLR7/8-inactive, but exhibited bacteriostatic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To investigate the mechanism of antibacterial activity of this new chemotype, a resistant strain of S. aureus was generated by serially passaging the organism in escalating doses of the most active analogue. A comparison of minimum inhibitory concentrations (MICs) of known bacteriostatic agents in wild-type and resistant strains indicates a novel mechanism of action. Structure–activity relationship studies have led to the identification of positions on the scaffold for additional structural modifications that should allow for the introduction of probes designed to examine cognate binding partners and molecular targets, while not significantly compromising antibacterial potency.     

In Memorium: Swamy Laxminarayan [1939–2005]

Swamy Narasimha Laxminarayan, known to his many friends and colleagues as Swamy, passed away on September 29, 2005. He was one of the most prominent biomedical engineers on the international scene, and contributed immensely to the globalization of this new field.     

HdrC2 from Acidithiobacillus ferrooxidans Owns Two Iron–Sulfur Binding Motifs But Binds Only One Variable Cluster Between [4Fe–4S] and [3Fe–4S]

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GS _n H species (n > 1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been confirmed to contain iron–sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe–4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx₂Cx₂Cx₃C) containing total of eight cysteine residues potentially for iron–sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster converted between [4Fe–4S] and [3Fe–4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe²⁺ condition loaded only one [4Fe–4S]⁺ with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe–4S]⁰ with spin S = 0; the HdrC2 in oxidation condition loaded only one [3Fe–4S]⁺ with spin S = 1/2. Molecular modeling results were also in line with the experiment results.     

3-β-d-erythrofuranosyl-1-phenylpyrazolo[3,4-b]quinoxaline,a new C-nucleoside analog

Dehydration of the hydroxyalkyl chain of 1-phenyl-3-(d-arabino-tetritol-1-yl)pyrazolo[3,4-b]quinoxaline gave the C-nucleoside 3-β-d-erythrofuranosyl-1-phenyl-pyrazolo[3,4-b]quinoxaline (2) in 82% yield. The structure, and the configuration at the anomeric carbon atom, of 2 were elucidated by periodate oxidation, c.d. and n.m.r. spectroscopy, and mass spectrometry. N.m.r.-spectral and c.d. studies revealed that, due to the large size of the heterocyclic base, compound 2 is formed by inversion in the configuration or C-1 of the side chain. The mechanism of the dehydrative cyclization with inversion is discussed.     

DNA–DNA cross-linking mediated by bifunctional [SalenAl] complex

The aluminum (III) complex [SalenAl^III]Cl (1), (Salen = (R,R)-N,N′-bis[5-methyl-3-(4-methylpiperazinyl)-salicylidene]-1,2-diphenylethanediamine) has been synthesized and characterized by elemental analysis, FT-IR, ¹H and ¹³C NMR measurements. The interaction of complex (1) with calf thymus (CT) DNA has been studied extensively by experimental techniques. Thermal denaturation study of DNA with (1) revealed the ΔT_m of 5 ± 0.2 °C. Viscosity and steady-state fluorescence measurements showed that the complex cross-links DNA and the metal center is interacting with DNA during the cross-linking. Also, the phenyl ring in the complex may intercalate between the base pairs of the DNA during the cross-linking. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of the metal complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (2.8 ± 0.2) × 10⁵ M^− 1. Further, time-resolved fluorescence experiments confirm the binding of (1) with DNA and its cross-linking nature. Aluminum ions shown to precipitate DNA completely above the pH 6.0, but no such precipitation was observed with complex (1). The DNA–DNA cross-linking mediated by (1) is further confirmed by gel electrophoresis.     

[^3H]Cortisol及[^3H]Dexamethasone在大鼠肝细胞膜上的特异结合位点

本研究应用［３Ｈ］ｃｏｒｔｉｓｏｌ和［３Ｈ］ｄｅｘａｍｅｔｈａｓｏｎｅ（ＤＥＸ）两种配基,观察到大鼠肝细胞膜上存在一类糖皮质激素（ＧＣ）特异结合位点。这些位点与ＧＣ的结合具有饱和性、高亲和力及低容量。其平衡解离常数（Ｋｄ）分别为１２．８４±６．５８ｎｍｏｌ／Ｌ和４０．２７±２３．４４ｎｍｏｌ／Ｌ;最大结合容量（Ｂｍａｘ）分别为２．５７±１．８４ｐｍｏｌ／ｍｇ蛋白质与０．６４±０．１８ｐｍｏｌ／ｍｇ蛋白质（ｃｏｒｔｉｓｏｌ,ｎ＝４;ＤＥＸ,ｎ＝３;±ＳＥ）。动力学实验数据所得的Ｋｄ值与Ｓｃａｔｃｈａｒｄ分析所得的Ｋｄ值结果基本一致。［３Ｈ］ｃｏｒｔｉｓｏｌ和［３Ｈ］ＤＥＸ饱和结合实验数据用Ｓｃａｔｃｈａｒｄ作图分析,均显示为直线。Ｈｉｌｌ系数则分别为０．９８８０和０．９９９０．竞争抑制实验结果表明,ｃｏｒｔｉｓｏｌ对［３Ｈ］ｃｏｒｔｉｓｏｌ的结合位点有高度特异性竞争,比其它几种类固醇（强的松、黄体酮、ＲＵ４８６、ＤＥＸ）的竞争力至少强４０倍以上．用放射自显影技术进行研究,也提供了［３Ｈ］ｃｏｒｔｉｓｏｌ特异结合银粒位于大鼠肝细胞膜上的依据。     

Intermittent Administration of Parathyroid Hormone [1–34] Prevents Particle-Induced Periprosthetic Osteolysis in a Rat Model

We examined whether intermittent administration of parathyroid hormone [1–34] (PTH[1–34]; 60 μg/kg/day) can prevent the negative effects of titanium (Ti) particles on implant fixation and periprosthetic osteolysis in a rat model. Eighteen adult male rats (12 weeks old, bones still growing) received intramedullary Ti implants in their bilateral femurs; 6 rats from the blank group received vehicle injections, and 12 rats from the control group and PTH treatment group received Ti particle injections at the time of operation and intra-articular injections 2 and 4 weeks postoperatively. Six of the rats that received Ti particles from the PTH group also received PTH[1–34] treatment. Six weeks postoperatively, all specimens were collected for assessment by X-ray, micro-CT, biomechanical, scanning electron microscopy (SEM), and dynamic histomorphometry. A lower BMD, BV/TV, Tb.N, maximal fixation strength, and mineral apposition rate were observed in the control group compared to the blank group, demonstrating that a periprosthetic osteolysis model had been successfully established. Administration of PTH[1–34] significantly increased the bone mineral density of the distal femur, BV/TV, Tb.N, Tb.Th, Tb.Sp, Con.D, SMI, and maximal fixation strength in the PTH group compared to that in the control group. SEM revealed higher bone–implant contact, thicker lamellar bone, and larger trabecular bone area in the PTH group than in the control group. A higher mineral apposition rate was observed in the PTH group compared to both the blank and control groups. These findings imply that intermittent administration of PTH[1–34] prevents periprosthetic osteolysis by promoting bone formation. The effects of PTH[1–34] were evaluated at a suprapharmacological dosage to the human equivalent in rats; therefore, additional studies are required to demonstrate its therapeutic potential in periprosthetic osteolysis.     

Microwave-assisted synthesis and structure–activity relationships of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives

We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the most efficient compounds to impair the locomotor activity in mice at the dose of 10 μmol/kg.     

[3H]norharman ([3H]β-carboline) binds reversibly and with high affinity to a specific binding site in rat liver

In addition to the known binding of norharman (NH) to monoamine oxidase (MAO) and benzodiazepine (BZ) binding sites (at M concentrations), a distinct class of high-affinity NH binding sites was discovered in rat brain (1,2). Investigations of several organs of the rat led to the discovery of high affinity binding sites in the liver, which successfully could be solubilized from P₂ membrane homogenate (0.25% w/v Triton X-100). Scatchard analysis revealed an apparent K_D value of 26±8 nM and a maximum number of binding sites of 11±3 pmol/mg protein (n=14). Association kinetics showed that equilibrium was nearly reached after two hours. Dissociaton was totally complete only after more than 16 hours. The MAO-inhibitors examined did not influence the binding characteristics. No displacement of specific binding could be found by haloperidol.     

In Memorium: Herman P. Schwan [1915–2005]

Herman P. Schwan [1915–2005] was a distinguished scientist and engineer, and a founding father of the field of biomedical engineering. A man of integrity, Schwan influenced the lives of many, including his wife and children, and his many students and colleagues. Active in science until nearly the end of his life, he will be very much missed by his family and many colleagues.     

EPR analysis of multiple forms of [4Fe–4S]3+ clusters in HiPIPs

The electron paramagnetic resonance (EPR) spectrum from the [4Fe–4S]³⁺ cluster in several high-potential iron–sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe–4S]³⁺ structure the (Fe–Fe)⁵⁺ mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe–4S]³⁺ paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe–4S]³⁺ model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.     

Desulfovibrio gigas ferredoxin II: redox structural modulation of the [3Fe–4S] cluster

Desulfovibrio gigas ferredoxin II (DgFdII) is a small protein with a polypeptide chain composed of 58 amino acids, containing one Fe₃S₄ cluster per monomer. Upon studying the redox cycle of this protein, we detected a stable intermediate (FdII_int) with four ¹H resonances at 24.1, 20.5, 20.8 and 13.7 ppm. The differences between FdII_ox and FdII_int were attributed to conformational changes resulting from the breaking/formation of an internal disulfide bridge. The same ¹H NMR methodology used to fully assign the three cysteinyl ligands of the [3Fe–4S] core in the oxidized state (DgFdII_ox) was used here for the assignment of the same three ligands in the intermediate state (DgFdII_int). The spin-coupling model used for the oxidized form of DgFdII where magnetic exchange coupling constants of around 300 cm⁻¹ and hyperfine coupling constants equal to 1 MHz for all the three iron centres were found, does not explain the isotropic shift temperature dependence for the three cysteinyl cluster ligands in DgFdII_int. This study, together with the spin delocalization mechanism proposed here for DgFdII_int, allows the detection of structural modifications at the [3Fe-4S] cluster in DgFdII_ox and DgFdII_int.     

PI3K Inhibition Augments the Therapeutic Efficacy of a 3a-aza-Cyclopenta[α]indene Derivative in Lung Cancer Cells

The synergistic targeting of DNA damage and DNA repair is a promising strategy for the development of new chemotherapeutic agents for human lung cancer. The DNA interstrand cross-linking agent BO-1509, a derivative of 3a-aza-cyclopenta[α]indene, was synthesized and combined with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 to treat human lung cancer cells. Our results showed that the BO-1509 and LY294002 combination synergistically killed lung cancer cells in culture and also suppressed the growth of lung cancer xenografts in mice, including those derived from gefitinib-resistant cells. We also found that LY294002 suppressed the induction of several DNA repair proteins by BO-1509 and inhibited the nuclear translocation of Rad51. On the basis of the results of the γH2AX foci formation assays, LY294002 apparently inhibited the repair of DNA damage that was induced by BO-1509. According to the complete blood profile, biochemical enzyme analysis, and histopathologic analysis of major organs, no apparent toxicity was observed in mice treated with BO-1509 alone or in combination with LY294002. Our results suggest that the combination of a DNA cross-linking agent with a PI3K inhibitor is a feasible strategy for the treatment of patients with lung cancer.     

Synthesis of Pyrrolo[2′,3′:4,5]Furo[3,2-c]Pyridine-2-Carboxylic Acids

1H-Pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylic acid (6a) and its 1-methyl (6b) and 1-benzyl (6c) derivatives were synthesized. 3-(5-Methoxycarbonyl-4H-furo[3,2-b]-pyrrole-2-yl)propenoic acid (1) was converted to the corresponding azide 2, which in turn was cyclized to give 3 by heating in diphenylether. The pyridone 3 obtained was aromatized with phosphorus oxychloride, then reduced with zinc in acetic acid to give methyl 1H-pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylate (5), which by hydrolysis gave the corresponding carboxylic acid 6a.