Characterization of Caenorhabditis elegans exonuclease-3 and evidence that a Mg2+-dependent variant exhibits a distinct mode of action on damaged DNA

The Caenorhabditis elegans genes, exo-3 and apn-1, encode the proteins EXO-3 and APN-1, belonging to the exo III and endo IV families of apurinic/apyrimidinic (AP) endonucleases/3'-diesterases, respectively. Homologues of EXO-3 and APN-1 in E. coli and yeast have been clearly documented to repair AP sites and DNA strand breaks with blocked 3' ends to prevent genomic instability. Herein, we purified the C. elegans EXO-3, expressed as a Gst-fusion protein in yeast, and demonstrated that it possesses strong AP endonuclease and 3'-diesterase activities. However, unlike the E. coli counterpart exonuclease III, EXO-3 shows no significant level of 3' --> 5' exonuclease activity following incision at AP sites. In addition, EXO-3 lacks the ability to directly incise DNA at the 5' side of various oxidatively damaged bases, as observed for the human counterpart Ape1, suggesting that C. elegans evolved a member with tailored functions. Importantly, a variant form of EXO-3, E68A, demonstrates altered magnesium-binding properties, and although the in vitro AP endonuclease is nearly fully recovered in the presence of MgCl2, the 3'-diesterase activity is reduced when compared to the native enzyme. We suggest that Glu68 plays a role in coordinating Mg2+ binding for the enzyme catalytic mechanism. Further analysis reveals that neither purified Gst-EXO-3 nor the E68A variant forms a readily detectable DNA-protein complex with an oligonucleotide substrate containing either an AP site or an alpha,beta-unsaturated aldehyde at its 3' end. However, if the reaction is conducted in the presence of crude extracts derived from either yeast or C. elegans embryos, only E68A forms a distinct slow migrating DNA-protein complex with each of the substrates, suggesting that Glu68 may be required to facilitate the release of EXO-3 from the incised DNA to allow entry of the remaining components of the base-excision repair pathway. Thus, the slow migrating DNA-protein complex formed by the E68A variant could be indicative of a stalled repair process with associated factor(s).     

Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct

Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.     

Effects of selenium on altered mechanical and electrical cardiac activities of diabetic rat

Since selenium compounds can restore some metabolic parameters and structural alterations of diabetic rat heart, we were tempted to investigate whether these beneficial effects extend to the diabetic rat cardiac dysfunctions. Diabetes was induced by streptozotocin (50mg/kg body weight) and rats were then treated with sodium selenite (5 micromol/kg body weight/day) for four weeks. Electrically stimulated isometric contraction and intracellular action potential in isolated papillary muscle strips and transient (I(to)) and steady state (I(ss)) outward K(+) currents in isolated cardiomyocytes were recorded. Sodium selenite treatment could reverse the prolongation in both action potential duration and twitch duration of the diabetic rats, and also cause significant increases in the diminished amplitudes of the two K(+) currents. Treatment of rats with sodium selenite also markedly increased the depressed acid-soluble sulfhydryl levels of the hearts. Our data suggest that the beneficial effects of sodium selenite treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to the restoration of the diminished K(+) currents, partially, related to the restoration of the cell glutathione redox cycle.     

In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.     

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.     

Mucoadhesive, thermosensitive, prolonged-release vaginal gel for clotrimazole:beta-cyclodextrin complex

The purpose of this study was to achieve a better therapeutic efficacy and patient compliance in the treatment for vaginitis. Clotrimazole (1%) has been formulated in a vaginal gel using the thermosensitive polymer Pluronic F127 (20%) together with mucoadhesive polymers such as Carbopol 934 and hydroxypropylmethylcellulose (0.2% for both). To increase its aqueous solubility, clotrimazole was incorporated as its inclusion complex with 1:1 molar ratio with beta-cyclodextrin. The inclusion complex was thoroughly characterized using various techniques, including 1H NMR spectroscopy, FT IR spectrophotometry, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and determination of stability constant (k(1:1)). The gelation temperature and rheological behavior of different formulations at varying temperatures were measured. In vitro release profiles of the gels were determined in pH 5.5 citrate buffer. It was observed that complexation with cyclodextrin slowed down the release of clotrimazole considerably. Carbopol 934, on the other hand, was found to interact with beta-cyclodextrin, inducing precipitation. As far as rheological properties are concerned, thermosensitive in situ gelling was obtained with formulations containing drug:cyclodextrin complex rather than with free drug. Thus, the optimum formulation for a controlled-release thermosensitive and mucoadhesive vaginal gel was determined to be clotrimazole:beta-cyclodextrin 1% with 0.2% hydroxypropylmethylcellulose in Pluronic F127 gel (20%) providing continuous and prolonged release of active material above MIC values.     

Selenium prevents diabetes-induced alterations in [Zn2+]i and metallothionein level of rat heart via restoration of cell redox cycle

Intracellular free zinc concentration ([Zn2+]i) is very important for cell functions, and its excessive accumulation is cytotoxic. [Zn2+]i can increase rapidly in cardiomyocytes because of mobilization of Zn2+ from intracellular stores by reactive oxygen species (ROS). Moreover, ROS have been proposed to contribute to direct and/or indirect damage to cardiomyocytes in diabetes. To address these hypotheses, we investigated how elevated [Zn2+]i in cardiomyocytes could contribute to diabetes-induced alterations in intracellular free calcium concentration ([Ca2+]i). We also investigated its relationship to the changes of metallothionein (MT) level of the heart. Cardiomyocytes from normal rats loaded with fura-2 were used to fluorometrically measure resting [Zn2+]i (0.52 +/- 0.06 nM) and [Ca2+]i (26.53 +/- 3.67 nM). Fluorescence quenching by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine was used to quantify [Zn2+]i. Our data showed that diabetic cardiomyocytes exhibited significantly increased [Zn2+]i (0.87 +/- 0.05 nM ) and [Ca2+]i (49.66 +/- 9.03 nM), decreased levels of MT and reduced glutathione, increased levels of lipid peroxidation and nitric oxide products, and decreased activities of superoxide dismutase, glutathione reductase, and glutathione peroxidase. Treatment (4 wk) of diabetic rats with sodium selenite (5 micromol.kg body wt(-1).day(-1)) prevented these defects induced by diabetes. A comparison of present data with previously observed beneficial effects of selenium treatment on diabetes-induced contractile dysfunction of the heart can suggest that an increase in [Zn2+]i may contribute to oxidant-induced alterations of excitation-contraction coupling in diabetes. In addition, we showed that oxidative stress is involved in the etiology of diabetes-induced downregulation of heart function via depressed endogenous antioxidant defense mechanisms.