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The effect of aging and treatment with acetyl-L-carnitine on the activity of cytochrome oxidase and adenine nucleotide translocase in rat heart mitochondria was studied. It was found that the activity of both these mitochondrial protein systems was reduced (by around 30%) in aged animals. Treatment of aged rats with acetyl-L-carnitine almost completely reversed this effect. Changes in the mitochondrial cardiolipin content appear to be responsible for these effects of acetyl-L-carnitine. Show less

Blocks of potential Z-DNA-forming (dA-dC)n.(dG-dT)n sequences are ubiquitous in eukaryotic genomes. We examined whether naturally occurring polyamines, putrescine, spermidine and spermine, could provoke the Z-DNA conformation in plasmids pDHf2 and pDHf14 with 23 and 60 bp inserts respectively of (dA-dC)n.(dG-dT)n sequences using an e.l.i.s.a. Spermidine and spermine could provoke Z-DNA conformation in these plasmids, but putrescine was ineffective. For pDHf2 and pDHf14, the concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 25 microM, whereas that of spermine was 16 microM. Polyamine structural specificity was evident in the ability of spermidine homologues to induce Z-DNA. Inorganic cations, Co(NH3)6(3+) and Ru(NH3)6(3+), were ineffective. Our experiments also showed increased binding of anti-DNA autoantibodies from lupus patients as well as autoimmune MRL-lpr/lpr mice to pDHf2 and pDHf14 in the presence of polyamines. These data demonstrate that small blocks of (dA-dC)n.(dG-dT)n sequences could assume the Z-DNA conformation in the presence of natural polyamines. Increased concentrations of polyamines in the sera of lupus patients might facilitate immune complex-formation involving circulating DNA and anti-Z-DNA antibodies. Show less

The interaction of calf-thymus DNA with cobalt-hexammine and cobalt-pentammine cations was investigated, in aqueous solution at pH 6-7 with cation/DNA(phosphate) molar ratios r = 1/80, 1/40, 1/20, 1/10, 1/4, 1/2 and 1, using Fourier Transform infrared (FTIR) difference spectroscopy. Correlations between spectral changes, DNA condensation and helical stabilization due to the cation interaction as well as conformational features are established. At a very low cation concentration (r = 1/80), the binding of cobalt-hexammine cation with DNA is through the H-bond formation between cation NH3 groups and the PO2 groups of the backbone, resulting in duplex stability. As the cation concentration increases, hydrogen bonding expands towards guanine N-7 and O-6 atoms. At r > 1/20, DNA condensation occurs with major reduction in the intensity of several DNA in-plane vibrations and that of the phosphate group. The cobalt-pentammine cation binding is via the PO2 groups (directly) at very low metal cation concentration (r = 1/80) and the guanine N-7 and the O-6 groups (indirectly) at higher ratios. At r > 1/10, DNA condensation begins with some degree of direct cation-base binding. No major conformational changes from the B-family structure were observed before and after DNA collapse, in the presence of cobalt-ammine cations. Show less

We have examined the effects of the cis-diammine and 1,2-diaminocyclohexane (dach) carrier ligands on cytotoxicity, platinum accumulation and efflux, platinum incorporation into DNA, cytotoxicity of Pt-DNA adducts, and repair of Pt-DNA adducts in the human ovarian carcinoma A2780 cell line, the human colon carcinoma HCT8 cell line, and their cis-diamminedichloroplatinum(II) (cisplatin)-resistant derivatives, A2780/DDP and HCT8/DDP. The A2780/DDP cell line was 7.7-fold resistant to cisplatin, and the HCT8/DDP cell line was 1.6-fold resistant to cisplatin compared to their parental cell lines. Both were considered as examples of acquired cisplatin resistance. The HCT8/S cell line was 4.6-fold resistant to cisplatin compared with the A2780/S cell line and was considered an example of intrinsic resistance. Decreased accumulation of cisplatin made a significant contribution to acquired cisplatin resistance in the A2780/DDP cell line, probably contributed to intrinsic resistance in the HCT8/S cell line, but made little or no contribution to acquired resistance in the HCT8/DDP cell line. Decreased cytotoxicity of Pt-DNA adducts made a major contribution to both acquired and intrinsic cisplatin resistance in all three cell lines. Increased repair activity made a significant contribution to the decreased cytotoxicity of Pt-DNA adducts in the HCT8/S cell line, a weak contribution in the A2780/DDP cell line, and no contribution in the HCT8/DDP cell line. Glutathione levels were elevated in all the cell lines with acquired and intrinsic resistance, but the increased glutathione levels were not associated with decreased incorporation of platinum into DNA. These data suggest that both decreased accumulation and increased repair contribute to cisplatin resistance to different degrees in these human carcinoma cell lines. In addition, mechanism(s) other than repair may contribute to the decreased cytotoxicity of cis-diammine-Pt-DNA adducts. Of the cells with acquired cisplatin resistance, the HCT8/DDP cell line showed no resistance to tetrachloro(trans-DL)1,2-diaminocyclohexaneplatinum(IV) (ormaplatin, formerly known as tetraplatin), while the A2780/DDP cell line was just as resistant to ormaplatin as to cisplatin. The intrinsically cisplatin-resistant HCT8/S cell line showed only partial cross-resistance to ormaplatin. The effects of the dach carrier ligand on both acquired and intrinsic resistance in these cell lines appeared to occur primarily at the level of cytotoxicity of dach-Pt adducts, but the differences in the cytotoxicity of cis-diammine-Pt and dach-Pt adducts could not be explained by differences in repair of those adducts.(ABSTRACT TRUNCATED AT 400 WORDS) Show less

Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R9) also bound specifically to TAR, and a mutant Tat protein that contained R9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K9) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine eta nitrogens and the epsilon nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures. Show less

The objective of this work has been to analyse the repartition of platinum (Pt) tissular levels within the tumour (T), the peritumoral adjacent non-tumoral area (P) and distant healthy tissue of the same anatomical zone (H) in oesophagus cancer. Forty-two biopsies (congruent to 5 mg) have been performed under endoscopy and after informed consent in 11 patients (mean age 61 yr, range 43-74) with squamous cell carcinoma of the oesophagus treated by the neoadjuvant chemotherapy protocol including cisplatin (100 mg/m2) and 5-FU (1 g/m2 x 5 days). Biopsies were done 34-36 h after cisplatin. Additional biopsies were obtained for histological controls. Pt was measured by flameless atomic absorption spectrometry. Considering Pt concentration in T, P and H there was no significant accumulation during repeated treatment (3 cycles). For all cycles, mean [S.D.] values (micrograms/g dry tissue) were 2.03 [2.39] for H, 2.75 [2.03] for P and 3.73 [2.3] for T (H vs. T, P = 0.006). In addition, Pt concentrations were found comparable between the upper and lower poles of the tumours (5 patients). Pt concentrations in T did not predict antitumour activity. These data complete the rather limited knowledge on tissular Pt levels in treated patients and suggest a decreasing gradient of Pt concentrations from tumour to healthy tissue in oesophagus cancer. Show less

The use of molecular biological methodologies has provided a greater understanding of the cytotoxic effects of cisplatin and the underlying mechanisms of tumour cell resistance. Resistance to cisplatin is often multifocal with plasma membrane, cytosolic and nuclear components. Cisplatin-DNA adducts appear to be recognised by specific damage recognition proteins. Proteins associated with the transport of platinum through plasma membranes and genes associated with cisplatin resistance appear to be close to being elucidated. Current Phase I and Phase II clinical trials with platinum-containing complexes largely focus on the 1,2 diaminocyclohexane (DACH) carrier ligand, the dicarboxylatocyclobutane leaving group and complexes which circumvent cisplatin resistance in murine leukaemia models. At present, the trials are at too early a stage to allow comment on their clinical utility and, consequently, the relevance of the murine leukaemia-based preclinical observations. On the horizon, orally active platinum (IV) ammine/amine dicarboxylate dichloride coordination complexes with preclinical toxicological profiles similar to carboplatin should enter clinical trial in the next year. Show less

Tumor-tissue and plasma concentrations of platinum were studied prospectively in two groups of eight patients who were suffering from advanced non-small-cell lung cancer. Treatments including two different schedules of cisplatin administration (25 vs 100 mg/m2 on day 1) were compared. At 30 min after the beginning of the cisplatin infusion, blood samples and bronchoscopically obtained biopsy specimens were taken for determinations of platinum concentrations by means of flameless atomic absorption spectrophotometry. The procedure did not induce any complication. Total plasma platinum concentrations at 30 min were significantly lower (P<0.01) in patients receiving 25 mg/m2 (0.49±0.23 μg Pt/ml) than in those receiving 100 mg/m2 (1.44±0.62 μg Pt/ml), whereas no significant difference was observed in tumor-tissue platinum concentrations (22.49±53.89 ng Pt/mg in patients receiving 25 mg/m2 vs 51.13±65.52 ng Pt/mg in those receiving 100 mg/m2). There was a weak correlation between simultaneous plasma and tumor-tissue platinum concentrations at 30 min. Tumor-tissue platinum concentrations seem to be poorly influenced by the cisplatin dose. This finding suggests a great interindividual variability of platinum tumor-diffusion properties in non-small-cell lung cancer. Show less

We studied the effects of hexammine and tris(ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH3)6(3+) provoked a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the psi-DNA form. In addition, we compared the efficacy of Rh(NH3)6(3+) and Rh(en)3(3+) to provoke the Z-DNA conformation in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC.poly(dG-m5dC). The concentrations of Rh(NH3)6(3+) and Rh(en)3(3+) at the midpoint B-DNA----Z-DNA transition of poly(dG-dC).poly(dG-dC) were 48 +/- 2 and 238 +/- 2 microM, respectively. The psi-DNA form of poly(dG-dC).poly(dG-dC) was stabilized at 500 microM Rh(NH3)6(3+). With poly(dG-m5dC).poly(dg-m5dC), both counterions provoked the Z-DNA form at approximately 5 microM and stabilized the polynucleotide in this form up to 1000 microM concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly(dG-dC).poly(dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects. Show less

SCANNING tunnelling microscopy (STM) has been used to map the surface topography of inorganic materials at the atomic level, and is potentially one of the most powerful techniques for probing biomolecular structure1–3. Recent STM studies of calf thymus DNA4,5and poly(rA) · poly(rU)5 have shown that the helical pitch and periodic alternation of major and minor grooves can be visualized and reliably measured. Here we present the first STM images of poly(dG-me5d) · poly(dG-me5dC) in the Z-form. Both the general appearance of the fibres and measurements of helical parameters are in good agreement with models derived from X-ray diffraction6–4. Show less

Hexammine cobalt(III) chloride (Co(NH3)6(3+) provokes a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC). The circular dichroism spectrum of psi-DNA is characterized by a manyfold increase of positive ellipticity in the range of 300-225 nm and the complete absence of a negative peak. In order to ascertain the helical handedness of psi-DNA, we used a recently developed enzyme immunoassay technique. This method consisted of treating the polynucleotides with Co(NH3)6(3+) to convert them to the Z- or psi-DNA forms and immobilizing these conformations on a microtiter plate. The plates were subsequently treated with a monoclonal anti-Z-DNA antibody Z22, alkaline phosphatase conjugated, affinity purified immunoglobulins, and the phosphatase substrate. The enzyme-substrate reaction was monitored by reading the absorbance at 405 nm with a microplate autoreader. The monoclonal anti-Z-DNA antibody had no reactivity to the B-DNA form, but bound strongly to both the Z- and psi-DNA forms, showing that Co(NH3)6(3+)-induced psi-DNA form of the polynucleotides exists in the left-handed Z-DNA conformation. Show less

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT). Show less

Specific high‐affinity binding sites for 125I‐α‐bungarotoxin and (−)‐[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I‐α‐bungarotoxin had similar K d values of 1.5 × 10−9 and 0.8 × 10−9 M for rat and locust preparations, respectively; the corresponding values for the (−)‐[3H]nicotine‐binding site were 9.3 × 10−9 and 1.7 × 10−7 M. Methyllycaconitine (MLA) potently inhibited 125I‐α‐bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (−)‐[3H]nicotine binding whereas (+)‐anatoxin‐a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)‐anatoxin‐a is a useful probe for the high‐affinity nicotine‐binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds α‐bungarotoxin. Show less

In the equilibrium between B-DNA and Z-DNA in poly(dC-dG), the [Co(NH3)6]3+ ion stabilizes the Z form 4 orders of magnitude more effectively than the Mg2+ ion. The structural basis of this difference is revealed in Z-DNA crystal structures of d(CpGpCpGpCpG) stabilized by either Na+/Mg2+ or Na+/Mg2+ plus [Co(NH3)6]3+. The crystals diffract X-rays to high resolution, and the structures were refined at 1.25 A. The [Co(NH3)6]3+ ion forms five hydrogen bonds onto the surface of Z-DNA, bonding to a guanine O6 and N7 as well as to a phosphate group in the ZII conformation. The Mg2+ ion binds through its hydration shell with up to three hydrogen bonds to guanine N7 and O6. Higher charge, specific fitting of more hydrogen bonds, and a more stable complex all contribute to the great effectiveness of [Co(NH3)6]3+ in stabilizing Z-DNA. Show less

Using a combination of spectroscopic techniques, quasi-elastic laser light scattering (QLS), and electron microscopy (EM), we have been able to show that the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC) is accompanied by extensive condensation of the DNA in both low and high ionic strength buffers. At low concentrations of NaCl (2 mM Na+), an intermediate rodlike form, which exhibits a circular dichroism (CD) spectrum characteristic of an equimolar mixture of B and Z forms, is observed. This is produced by the orderly self-association of about four molecules of the polymer after prolonged incubation of a concentrated solution at 4 degrees C. On addition of 5 microM Co(NH3)63+, the CD spectrum of the intermediate changes to that of the Z form, which is visualized as a dense population of discrete toroids on an EM grid stained with uranyl acetate. On the other hand, addition of NaCl to a solution of poly(dG-m5dC) X poly(dG-m5dC) in the absence of any multivalent ion condenses the polymer to toroidal structures at the midpoint (0.75 M NaCl) of the B to Z transition. Further addition of NaCl unfolds these toroids to rodlike structures, which show characteristic Z-form CD spectra. These results show that Z DNA can take up a variety of tertiary structural forms and indicate that its inverted CD spectrum is due to its left-handed helical sense rather than to differential scattering artifacts. Show less