Ischemic tolerance - blessing or curse.

Application of knowledge about ischemic tolerance to clinic requires the solid understanding of mechanism of creation of this phenomenon. This review summarizes research that has been carried out in many laboratories over a long period of time, but the main focus will be on own experimental research. The main emphasis is devoted to the possibility of preparing full tolerance in the donor's body and its transfer to the patient in the form of activated blood plasma. Such plasma could be administered as soon as the patient is transported to the hospital and would take effect immediately after administration to the patient's bloodstream. One chapter is also devoted to anticonditioning, i.e. the possibility of preventing the activation of tolerance. Anticonditioning could be used to treat oncologic patients. We expect that this method could increase effectiveness of cancer treatment. Cross-tolerance with a wide range of diverse stressors gives us the courage to assume that activated plasma can significantly help with a wide range of pathological events.

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs.

BACKGROUND: Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum. METHODS: Inhibitory neuromuscular transmission was investigated using in vitro ileal longitudinal muscle myenteric plexus (LMMP) preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 weeks postnatal) guinea pigs. KEY RESULTS: Amperometric measurements of nicotine-induced nitric oxide (NO) release (measured as an oxidation current) from myenteric ganglia revealed larger currents in neonatal (379 +/- 24 pA) vs adult (119 +/- 39 pA, P < 0.05) tissues. Nicotine-induced oxidation currents were blocked by the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine (NLA, 100 micromol L(-1)). Nicotine-induced, NLA-sensitive oxidation currents could be detected in the tertiary plexus of neonatal but not adult tissues. Immunohistochemistry demonstrated stronger NOS immunoreactivity in neonatal compared with adult myenteric ganglia. Western blot studies revealed higher levels of NOS in neonatal compared with adult LMMP. Cell counts revealed that the total number of myenteric neurons in the small intestine was greater in adults than in neonatal guinea pigs, however, the ratio of NOS : Calbindin neurons was significantly higher in neonatal compared with adult tissues. CONCLUSIONS & INFERENCES: Nitric oxide signaling to the longitudinal muscle is stronger in neonatal compared with adult guinea pig ileum. Nitric oxide synthase-containing neurons are diluted postnatally by cholinergic and other, as yet unidentified neuronal subtypes.

Ischemic postconditioning in the rat hippocampus: mapping of proteins involved in reversal of delayed neuronal death.

In this study, transient forebrain ischemia was induced in male Wistar rats with subsequent 3 days of reperfusion (ischemia/reperfusion group) or 2 days of reperfusion followed by 5 min ischemia and another 1 day of reperfusion (postconditioning group) to assess an effect of delayed postconditioning applied two days after a previous lethal ischemic attack. We have examined immunoreactivity of antioxidant enzymes (MnSOD, CuZnSOD) and proteins related to apoptosis development (Bcl-2, Bax). Results of microdensitometric measurements from the vulnerable hippocampal CA1 region and relatively resistant dentate gyrus were compared to sham controls and identically, results of postconditioning group were compared to ischemic one. Our findings show protective effects of postconditioning in both brain regions examined, include increased expression of antioxidant enzymes, mainly CuZnSOD, what can be demonstrated by microdensitometric results: CuZnSOD density after ischemia and reperfusion was 6261.5 +/- 411.35; after postconditioning 9746.6 +/- 584.55. In addition, postconditioning prevents an excessive ischemia-induced increase of pro-apoptotic protein Bax (Bax density after ischemia and reperfusion was 3462.51 +/- 321.66; after postconditioning 1766.89 +/- 255.63).

Postnatal downregulation of inhibitory neuromuscular transmission to the longitudinal muscle of the guinea pig ileum.

Neuromuscular transmission is crucial for normal gut motility but little is known about its postnatal maturation. This study investigated excitatory/inhibitory neuromuscular transmission in vitro using ileal nerve-muscle preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 months postnatal) guinea pigs. In tissues from neonates and adults, nicotine (0.3-30 micromol L(-1)) contracted longitudinal muscle preparations in a tetrodotoxin (TTX) (0.3 micromol L(-1))-sensitive manner. The muscarinic receptor antagonist, scopolamine (1 micromol L(-1)), reduced substantially nicotine-induced contractions in neonatal tissues but not adult tissues. In the presence of N(omega)-nitro-l-arginine (NLA, 100 micromol L(-1)) to block nitric oxide (NO) mediated inhibitory neuromuscular transmission, scopolamine-resistant nicotine-induced contractions were revealed in neonatal tissues. NLA enhanced the nicotine-induced contractions in neonatal but not in adult tissues. Electrical field stimulation (20 V; 0.3 ms; 5-25 Hz, scopolamine 1 micromol L(-1) present) caused NLA and TTX-sensitive longitudinal muscle relaxations. Frequency-response curves in neonatal tissues were left-shifted compared with those obtained in adult tissues. Immunohistochemical studies revealed that NO synthase (NOS)-immunoreactivity (ir) was present in nerve fibres supplying the longitudinal muscle in neonatal and adult tissues. However, quantitative studies demonstrated that fluorescence intensity of NOS-ir nerve fibres was higher in neonatal than adult tissues. Nerve fibres containing substance P were abundant in longitudinal muscle in adult but not in neonatal tissues. Inhibitory neuromuscular transmission is relatively more effective in the neonatal guinea pig small intestine. Delayed maturation of excitatory motor pathways might contribute to paediatric motility disturbances.

[Simple bony fusion or instrumented hemivertebra excision in the surgical treatment of congenital scoliosis]. / Prostá kostená fúze versus instrumentovaná hemivertebrektomie v operacní lécbe kongenitálních skolióz.

PURPOSE OF THE STUDY: In a retrospective study, to analyze long-term radiographic results of two surgical procedures used to treat congenital scoliosis. MATERIAL AND METHODS: A total of 685 patients with congenital scoliosis were treated at the Department of Orthopaedic Surgery, Bohunice Teaching Hospital in Brno, between 1976 and 2007. Of these, 102 patients, with an average age of 6.6 years at the time of surgery, were treated by simple bony fusion, and 22 children, with an average age of 10.2 years, underwent instrumented hemivertebra excision via simultaneous anterior and posterior exposures involving fixation with cannulated compression screws and a wire loop. The follow-up periods for the former and latter groups were 14.2 and 12.1 years, respectively. RESULTS: In the patients treated by simple bony fusion, the mean correction rate was 22.1 %, with Cobb angle values averaging from 44.2 degrees pre-operatively to 38 degrees post-operatively; the correction loss was 3.9 degrees at the last follow up. In the patients with hemivertebra excision, the mean correction rate was 61 %, with pre- and post-operative values of 51.3 degrees and 20.3 degrees , respectively, and a correction loss of 1.1 degrees at the last follow-up. DISCUSSION: The early detection of a deformity and simple bony fusion in low-magnitude curves can prevent progression of scoliosis and allows for maintenance of a compensated spine. Hemivertebra excision with compression instrumentation results in a better surgical correction of the deformity. The average 61 % correction rate achieved in our patients is in agreement with the results reported by authors using the same surgical technique, as well as with the results of posterior hemivertebra resection. The best correction, 78 %, has been achieved with surgery at a very young age. Complications associated with the two techniques are rare. CONCLUSIONS: Congenital scoliosis due to failure off either formation or segmentation is indicated for surgical treatment at young age. Its early detection and subsequent surgical treatment at young age. Its early detection and subsequent surgical correction of the curve result in a long-term maintenance of a compensated spine. Instrumented hemivertebra excision provides the highest rate of correction, particularly if carried out before 3 years of age.

Calpain-induced proteolysis after transient global cerebral ischemia and ischemic tolerance in a rat model.

The activation of the [Ca(2+)]-dependent cysteine protease calpain plays an important role in ischemic injury. Here, the levels of two calpain-specific substrates, p35 protein and eukaryotic initiation factor 4G (eIF4G), as well as its physiological regulator calpastatin, were investigated in a rat model of transient global cerebral ischemia with or without ischemic tolerance (IT). Extracts of the cerebral cortex, whole hippocampus and hippocampal subregions after 30 min of ischemia and different reperfusion times (30 min and 4 h) were used. In rats without IT, the p35 levels slightly decreased after ischemia or reperfusion, whereas the levels of p25 (the truncated form of p35) were much higher than those in sham control rats after ischemia and remained elevated during reperfusion. The eIF4G levels deeply diminished after reperfusion and the decrease was significantly greater in CA1 and the rest of the hippocampus than in the cortex. By contrast, the calpastatin levels did not significantly decrease during ischemia or early reperfusion, but were upregulated after 4 h of reperfusion in the cortex. Although IT did not promote significant changes in p35 and p25 levels, it induced a slight increase in calpastatin and eIF4G levels in the hippocampal subregions after 4 h of reperfusion.

The effects of normovolemic hemodilution on protein synthesis recovery following postischemic reperfusion in the rat brain.

Normovolemic hemodilution is a possible way to improve the brain recovery after ischemia and reperfusion. Therefore we have decided to examine how this process may affect the post-ischemic protein synthesis machinery. We analysed rat brains after 4-vessel-occlusion and different time intervals of reperfusion using normovolemic hemodilution. We achieved an important increase of [4,5-3H]leucine incorporation into polypeptides in vitro in the rat brain neocortex 30 minutes after ischemia, but concurrently there was no significant change in the hippocampus and striatum. By extending the time course of reperfusion we did not observe any important deviation of in vitro [4,5-3H]leucine incorporation in the studied brain areas. Thus, although hemodilution increased protein synthesis in selective vulnerable regions after ischemia, this improvement is not of significant importance.

The protective effect of aminoguanidine on cerebral ischemic damage in the rat brain.

The NADPH-diaphorase (NADPH-d) histochemical technique is commonly used to localize the nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) in neural tissue. The expression of inducible nitric oxide synthase (iNOS) is induced in the late stage of cerebral ischemia, and NO produced by iNOS contributes to the delay in recovery from brain neuronal damage. The present study was performed to investigate whether the increase in nitric oxide production via inducible nitric oxide synthase was suppressed by the administration of aminoguanidine, a selective iNOS inhibitor, as it follows a decrease of NADPH-diaphorase activity (a marker for NOS) after four-vessel occlusion used as an ischemic model. The administration of aminoguanidine (100 mg/kg i.p., twice per day up to 3 days immediately after the ischemic insult) reduced the number of NADPH-diaphorase positive cells to control levels. Our results indicated that aminoguanidine suppressed NADPH-diaphorase activity, and also decreased the number of NADPH-diaphorase positive cells in the CA1 region of the hippocampus following ischemic brain injury.

The effect of preconditioning on the iron deposition after transient forebrain ischemia in rat brain.

In this study we investigated iron deposition in the hippocampus CA1 area and the corpus striatum pars dorsolateralis in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by four-vessel occlusion for 5-min as ischemic preconditioning. Two days after the preconditioning or the sham operation, a second ischemia was induced for 20-min. With the use of iron histochemistry, regional changes were examined after 2 to 8 weeks of recirculation following the 20-min ischemia with or without preconditioning. Perl's reaction with DAB intensification demonstrated iron deposits in the CA1 area and in the corpus striatum pars dorsolateralis after 2 weeks of recirculation. These iron deposits gradually increased in density and formed clusters by the 8th week. When the rats were exposed to 5-min ischemia 2 days before lethal 20-min ischemia, the deposition of iron in the CA1 region of the hippocampus and also in the corpus striatum pars dorsolateralis was decreased and produced a minimal number of iron-containing cells between the second and the 8th week of recirculation. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 region induced by 20-min ischemia.

Does phosphorylation of eukaryotic elongation factor eEF2 regulate protein synthesis in ischemic preconditioning?

Ischemia/reperfusion-associated translation inhibition in the hippocampus is attenuated significantly at reinitiation and elongation steps by ischemic preconditioning (Burda et al. [2003] Neurochem. Res. 28:1237-1243). To address potential regulation of the elongation step by changes in eukaryotic elongation factor 2 (eEF2) phosphorylation with and without acquired ischemic tolerance (IT), Wistar rats were preconditioned by 5-min sublethal ischemia and 2 days later, 30-min lethal ischemia was induced. Given the important role that oxidative stress plays in the ischemic process, eEF2 phosphorylation was also studied in a model of oxidative stress in vitro. Three blocks of our results support a lack of correlation between eEF2 phosphorylation status and protein synthesis rate. First, eEF2 was dephosphorylated significantly (activated) after transient cerebral ischemia in rats with and without IT or H2O2-treated cells; however, protein synthesis was significantly inhibited under these three conditions. Second, after 30-min reperfusion, the protein synthesis rate was maintained below control levels in cortex and hippocampus of rats without IT. Eukaryotic EF2 phosphorylated levels were notably low only in the cortex, whereas levels in the hippocampus were close to that of sham controls. In rats with IT, protein synthesis was virtually restored in both brain regions, but phosphorylated eEF2 levels were even higher than in rats without IT. Third, after 4-hr reperfusion, the protein synthesis rate in cortex and hippocampus was observed to be below sham control values in rats with and without IT. Conversely, phosphorylated eEF2 levels were below sham control in rats with IT and reached sham control values in rats without IT.

Effect of Tanakan on postischemic activity of protein synthesis machinery in the rat brain.

A growing body of evidence supports the role of free radicals in triggering the functional and metabolic disturbances following transient cerebral ischemia. This study was designed to evaluate whether the extent of reperfusion-induced inhibition of protein synthesis initiation as well as tissue injury can be reduced by Tanakan (Ginkgo biloba extract, EGb 761) (Beaufour-Ipsen Industrie). Rats received Tanakan in the dose of 40 mg/kg/day for 7 days before surgical intervention. Transient forebrain ischemia was induced by 4-vessel occlusion. Rats were subjected to 20 min of ischemia followed by 30 min, 4 h or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. The protective effect was observed, concerning both the protein synthesis and the number of surviving neurons, in the Tanakan-treated groups. Tanakan significantly reduced the ischemia/reperfusion-induced inhibition of translation in the neocortex as well as in the highly sensitive hippocampus. Our results indicate that free radicals play an important role in the development of reperfusion-induced injury, and the treatment of ischemic and reperfused brain with free radical scavengers may reduce the severity of reperfusion damage.

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain.

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30 min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70(S6K)) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4--6 h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4 h of reperfusion only in the cortex. Conversely, p70(S6K) activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2 alpha, eIF2B, eIF4G and p70(S6K) are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Ischemia-induced inhibition of the initiation factor 2alpha phosphatase activity in the rat brain.

Rats were subjected to the standard four-vessel occlusion model of brain transient ischemia for 30 min. Following different recirculation periods, the level of phosphorylation of the initiation factor 2 subunit alpha (eIF2alpha) and the eIF2alpha kinase/s and phosphatase/s activity were determined. eIF2alpha phosphorylation significantly increased very early during reperfusion (10-30 min), recovering at 4 h of reperfusion. Activation of any eIF2alpha kinases studied during ischemia or reperfusion was not noted. Conversely, eIF2alpha phosphatase activity significantly decreased at 10-15 min of reperfusion, reaching values even higher than in controls at 2-4 h of reperfusion. Our results support the hypothesis that the reperfusion-induced phosphorylated eIF2alpha changes are at least a result of the transiently eIF2alpha phosphatase inhibition.

The interactions of square platinum(II) complexes with guanine and adenine: a quantum-chemical ab initio study of metalated tautomeric forms.

The influence of binding of square planar platinum complexes on tautomeric equilibria of the DNA bases guanine and adenine was investigated using the density functional B3LYP method. Neutral trans-dichloro(amine)-, +1 charged chloro(diamine)-, and +2 charged triamine-platinum(II) species were chosen for coordination to bases. Only the N7 interaction site of the bases was considered. The calculations demonstrate that the neutral platinum adduct does not change the tautomeric equilibria of the bases. Furthermore, N7 binding of the neutral Pt adduct moderately reduces the probability of protonation of the N1 position of adenine. Larger effects can be observed for +1 and mainly +2 adducts, but these can be rationalized by electrostatic effects. Since the electrostatic effects are expected to be efficiently compensated for by a charged backbone of DNA and counterions in a polar solvent, no dramatic increase in mispair formation is predicted for Pt(II) adducts, which is in agreement with experiment. The interaction energies between Pt adducts and the nucleobases were also evaluated. These interaction energies range from ca. 210 kJ/mol for neutral adducts, interacting with both bases and their tautomers, up to 500 kJ/mol for the +2 charged adducts, interacting with the keto-guanine tautomer and the anti-imino-adenine tautomer. The surprisingly large interaction energy for the latter structure is due to the strong H-bond between the NH3 ligand group of the metal adduct and the N6 nitrogen atom of the base.

Metal ions in non-complementary DNA base pairs: an ab initio study of Cu(I), Ag(I), and Au(I) complexes with the cytosine-adenine base pair.

Ab initio calculations have been carried out to characterize the structure and energetics of a silver(I) complex with the cytosine-adenine DNA base pair and an aqua ligand in the coordination sphere of Ag. In addition, we have also studied analogous complexes with Cu(I) and Au(I), and structures in which adenine has been replaced by purine in order to investigate the structural role of the adenine amino group. The calculations revealed that all metal-modified structures are dominated by the metal-base interactions, while the water-metal ion interaction and many-body interligand repulsion are less important contributions. Nevertheless, the structural role of the water molecule in the complex is quite apparent and in agreement with an earlier crystallographic study. The metal-modified base pairs exhibit large conformational flexibility toward out-of-plane motions (propeller twist and buckle), comparable or, in some cases, even larger than that observed in the base pairs without metal ions. All structures have been optimized within the Hartree-Fock approximation, while interaction energies were evaluated with the inclusion of electron correlation.

Interactions of hydrated IIa and IIb group metal cations with thioguanine-cytosine DNA base pair: Ab initio and density functional theory investigation of polarization effects, differences among cations, and flexibility of the cation hydration shell.

The structures and energies of the thioguanine-cytosine Watson-Crick (thioGC WC) base pair interacting with hydrated IIa (Mg2+, Ca2+, Ba2+) and IIb group (Zn2+, Cd2+, Hg2+) cations have been studied using ab initio techniques. Furthermore, complexes between guanine and thioguanine with hydrated cations have been characterized assuming various structures of the hydration shells. The complexes of the thioGC WC base pair with hydrated cations have similar properties as the previously studied GC WC base pair. There is substantial polarization stabilization of the base pairing due to cation binding which amounts to 7 - 11 kcal/mol. Soft Cd2+ and Hg2+ cations have a uniquely strong interaction with the thiogroup and induce substantial nonplanarity of the pairing. The thiogroup tends to reduce the number of water molecules in the first hydration shell of the cation. All complexes were optimized within the Hartree-Fock (HF) approximation while their energetics has been evaluated using the second-order Moller-Plesset perturbational method (MP2). All interaction energy evaluations and a substantial portion of the optimizations of the hydrated cation-(thio)guanine complexes have been repeated using Becke-3LYP Density Functional Theory method. All three approximations used (HF, Becke-3LYP, and MP2) give qualitatively the same results for the present cationic complexes. The results demonstrate specific differences among the cations and provide a set of reference structures and energies for verification and/or parametrization of empirical potentials and other theoretical methods.

The intraischemic and early reperfusion changes of protein synthesis in the rat brain. eIF-2 alpha kinase activity and role of initiation factors eIF-2 alpha and eIF-4E.

Rats were subjected to the standard four-vessel occlusion model of transient cerebral ischemia (vertebral and carotid arteries). The effects of normothermic ischemia (37 degrees C) followed or not by 30-minute reperfusion, as well as 30-minute postdecapitative ischemia, on translational rates were examined. Protein synthesis rate, as measured in a cell-free system, was significantly inhibited in ischemic rats, and the extent of inhibition strongly depended on duration and temperature, and less on the model of ischemia used. The ability of reinitiation in vitro (by using aurintricarboxylic acid) decreased after ischemia, suggesting a failure in the synthetic machinery at the initiation level. Eukaryotic initiation factor 2 (eIF-2) presented almost basal activity and levels after 30-minute normothermic ischemia, and the amount of phosphorylated eIF-2 alpha in these samples, as well as in sham-control samples, was undetectable. The decrease in the levels of phosphorylated initiation factor 4E (eIF-4E) after 30-minute ischemia (from 32% to 16%) could explain, at least partially, the impairment of initiation during transient cerebral ischemia. After reperfusion, eIF-4E phosphorylation was almost completely restored to basal levels (29%), whereas the level of phosphorylated eIF-2 alpha was higher (13%) than in controls and ischemic samples (both less than 2%). eIF-2 alpha kinase activity in vitro as measured by phosphorylation of endogenous eIF-2 in the presence of ATP/Mg2+, was higher in ischemic samples (8%) than in controls (4%). It seems probable that the failure of the kinase in phosphorylating eIF-2 in vivo during ischemia is due to the depletion of ATP stores. The levels of the double-stranded activated eIF-2 alpha kinase were slightly higher in ischemic animals than in controls. Our results suggest that the modulation of eIF-4E phosphorylation could be implicated in the regulation of translation during ischemia. On the contrary, phosphorylation of eIF-2 alpha, by an eIF-2 alpha kinase already activated during ischemia, represents a plausible mechanism for explaining the inhibition of translation during reperfusion.

Hydrogen-bonded trimers of DNA bases and their interaction with metal cations: ab initio quantum-chemical and empirical potential study.

Neutral (G.GC, A.AT, G.AT, T.AT, and C(imino).GC) and protonated (CH+.GC and AH+.GC) hydrogen-bonded trimers of nucleic acid bases were characterized by ab initio methods with the inclusion of electron correlation. In addition, the influence of metal cations on the third-strand binding in Purine-Purine-Pyrimidine (Pu.PuPy) reverse-Hoogsteen triplets has been studied. The ab initio calculations were compared with those from recently introduced force fields (AMBER4.1, CHARMM23, and CFF95). The three-body term in neutral trimers is mostly negligible, and the use of empirical potentials is justified. The only exception is the neutral G.GC Hoogsteen trimer with a three-body term of -4 kcal/mol. Protonated trimers are stabilized by molecular ion-molecular dipole attraction and the interaction within the complex is nonadditive, with the three-body term on the order of -3 kcal/mol. There is a significant induction interaction between the third-strand protonated base and guanine. The calculations indicate an enhancement of the third-strand binding in the G.GC reverse-Hoogsteen trimer due to-metal cation coordination to the N7/O6 position of the third-strand guanine. Interactions between metal cations and complexes of DNA bases are in general highly non-additive; the three-body term is above-10 kcal/mol in a complex of a divalent cation (Ca2+) with the GG reverse-Hoogsteen pair. The pairwise additive empirical potentials qualitatively underestimate the binding energy between cation and base.

Structural and functional heterogeneity of integrated recombinant AAV genomes.

Adeno-associated Virus (AAV) has emerged as a promising vector for gene therapy because of its ability to generate high titer recombinant stocks and the potential for site specific integration. However, much of the current knowledge regarding the transduction and integration biology of this virus is based on studies evaluating wild type AAV or recombinant AAV which was unknowingly contaminated with wild type virus. Given the fact that recombinant AAV is replication incompetent, by virtue of deleted viral rep proteins responsible for site specific integration of the wild type virus, the integration process for recombinant AAV may likely be different from its wild type counterpart. To this end, the present study has attempted to elucidate the proviral structure of stably integrated recombinant AAV genomes harboring the alkaline phosphatase reporter gene in 293 and IB3 cell lines. Initial studies attempted to functionally characterize differences in proviral genomes using mobilization assays with assessed both liberated episomal recombinant AAV and infectious virus following transfection with Rep/Cap containing plasmids and/or infection with recombinant adenovirus (Ad). Using Southern and polymerase chain reaction (PCR) analysis, evaluation of genomic DNA from AAV clonal cell lines indicated that head to tail orientations of ITRs were absolutely required for excision of episomal genomes and rescue of infectious recombinant virus. Furthermore, mobilization of proviral DNA could be achieved in the presence of exogenous Rep/Cap without adenovirus, while mobilization of infectious recombinant virus required the addition of both Rep/Cap and Ad. Genomic Southerns suggest that two predominant proviral structures exist for recombinant AAV including head to head and tail to head duplex genomes. A third class of monomer proviral genomes with head to tail oriented ITRs was also observed. No evidence for tail to tail ITR oriented proviral genomes was detected in any of the clonal cell lines. Such findings have begun to lay the foundation for a clearer understanding of the mechanism of recombinant AAV integration and how this process differs from wild type AAV.