Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.

Relation between the rapid focal activation in the pulmonary vein and the maintenance of paroxysmal atrial fibrillation.

This case report describes a patient with drug refractory paroxysmal atrial fibrillation (PAF). Rapid focal activations with multiple sharp spikes were continuously identified inside the left superior pulmonary vein (PV) during sustained AF. Among seven episodes of AF, cessation of rapid focal activations coincided with the conversion of AF to flutter (n = 4) or immediate AF termination (n = 3). Guided by sharp spikes in the PV, abrupt termination of AF occurred during radiofrequency energy application. Conclusively, rapid focal activations inside the PV are critical in AF maintenance. Cessation of these rapid focal activations underlies the mechanism by which AF converts to flutter.

Significance of glutamate and dopamine neurons in the ventral pallidum in the expression of behavioral sensitization to amphetamine.

To explore the significance of ventral pallidum (VP) during the amphetamine sensitization, we first investigated if there are neurochemical alterations in the VP during amphetamine withdrawal period. Chronic amphetamine-treated (5 mg/kg x 14 days) rats displayed an apparent locomotion sensitization as compared with saline controls when challenged with 2 mg/kg amphetamine at withdrawal days 10-14. A microdialysis analysis revealed that output of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the VP of amphetamine-sensitized rats increased approximately two-fold as compared to controls at both pre- and post-amphetamine challenge period. On the other hand, the in vivo glutamate output in the VP increased upon amphetamine challenge in the behaviorally sensitized rats, but not in the controls. To evaluate if drug manipulation in the VP would affect the behavioral sensitization, we treated both groups of rats with NMDA receptor antagonist, MK-801 (5 microg/microl for 5 days; bilateral) in the VP during withdrawal days 6-10. Animals were challenged with 2 mg/kg amphetamine at withdrawal day 11. The behavioral profile exhibited that MK-801 pre-treatment significantly blocked the locomotion hyperactivity in amphetamine-sensitized rats. Taken together, the current results suggest that the excitatory amino acid in the VP plays a significant role during the expression of behavioral sensitization to amphetamine.

Differential effects of endomorphin-1 and -2 on amphetamine sensitization: neurochemical and behavioral aspects.

Mu-opioid receptors are known to modulate mesolimbic dopaminergic activity in the ventral tegmental area via disinhibition of GABA-containing neurons. Recently, two novel tetrapeptides, endomorphin-1 and endomorphin-2, were identified in the mammalian brain and reported to have high binding affinities toward mu-opioid receptors. To determine if endomorphins would modulate the development of amphetamine sensitization, we administered endomorphins locally into the rat brain followed by behavioral and neurochemical examinations. The results indicate that rats pretreated with endomorphin-1 or -2 (5 microg per side for 7 days) in the ventral tegmental area developed locomotor sensitization to the challenge injection of amphetamine (1 mg/kg). On the other hand, when endomorphins were given in the lateral ventricle (20 microg for 5 days) of amphetamine-sensitized rats (5 mg/kg x 14 days) during the withdrawal period (w5-w9), neither peptide had a modulatory effect on locomotor sensitization. Biochemical analyses revealed that treatment with endomorphins in the ventral tegmental area significantly increased the levels of glutamate in the medial prefrontal cortex and ventral and dorsal striatum to levels comparable to those observed in the amphetamine-sensitized rats. In the same animals, endomorphins also caused decreases in the levels of serotonin and its metabolite, 5-hydroxyindoleacetic acid, in the medial prefrontal cortex. Interestingly, although there was no behavioral significance, endomorphin-1 treatment in the lateral ventricle of control and amphetamine-sensitized rats during withdrawal resulted in decreases of GABA, aspartate, dopamine, and its metabolite 3,4-dihydroxyphenylacetic acid in the ventral striatum. We conclude that endomorphins, by stimulating the mu-opioid receptors in the ventral tegmental area, could sensitize the behavioral response to amphetamine. The results also demonstrate that there are differential responses between endomorphin-1 and -2 on behavioral amphetamine sensitization and the underlying neurochemical substrates.

Systemic administration of naked DNA: gene transfer to skeletal muscle.

Skeletal muscle is a promising target tissue for the gene therapy of both muscle and non-muscle disorders. Gene transfer into muscle tissue can produce a variety of physiologically active proteins and may ultimately be applied to the treatment of many diseases. A variety of methods have been studied to transfer genes into skeletal muscle, including viral and non-viral vectors. In this review, we discuss recent developments in the non-viral delivery of genes to muscles.

Targeted delivery of plasmid DNA to myogenic cells via transferrin-conjugated peptide nucleic acid.

We describe a novel approach to conjugate a targeting ligand to plasmid DNA without affecting either its supercoiled conformation or its ability to be efficiently transcribed. A 14-mer peptide nucleic acid (PNA) containing lysine and cysteine on each end was designed to target to a unique sequence located at the antibiotic resistance gene of the plasmid. The binding of PNA to the plasmid was found to be dose-dependent and sequence-specific and not to change the conformation of the plasmid. Transferrin (Tf) was conjugated with PNA via a reversible disulfide bond using N-succinimidyl-3-(2-pyridyldithio)propionate. Tf-PNA retained the ability to the plasmid in a sequence-specific manner. The efficiency of this bioconjugate for delivering plasmid was examined in cultured myoblasts and myotubes. Naked DNA and Tf-PNA/DNA showed no transfection activity in either myoblasts or myotubes. Polyethyleneimine (PEI) is required for significant increase of the transfection efficiency. At N:P ratio of 5, Tf-PNA enhanced gene transfection about fourfold over that of the DNA/PEI complex in both myoblasts and myotubes. This enhancement could be inhibited by excess free Tf, indicating that the enhancement of transfection was through Tf-mediated endocytosis. These findings suggest that this targeting system may have the potential for gene transfer to myogenic cells in vivo.

Significantly higher levels of oxidized LDL autoantibody in coronary artery disease patients.

BACKGROUND: Increasing evidence shows that oxidized low-density lipoprotein (ox-LDL) might play an important role in the pathogenesis of atherosclerosis. Ox-LDL is immunogenic and induces an autoantibody, which we used as a tool for measuring the content of ox-LDL in vivo. METHODS: Patients who were admitted for diagnostic cardiac catheterization for typical or atypical angina pectoris were enrolled in this study. After fasting for 12 hours, a venous blood sample was drawn from the antecubital vein for testing triglyceride, total cholesterol, LDL cholesterol, high-density lipoprotein (HDL) cholesterol, and ox-LDL autoantibody. The ox-LDL autoantibody was quantified using an enzyme linked immunosorbent assay. All patients underwent coronary angiography. Those who had more than 50% angiographic coronary luminal stenosis, were grouped into the coronary artery disease (CAD) group. RESULTS: Sixty-four patients were enrolled in the study (male/female = 46/18; mean +/- standard deviation, age, 64 +/- 9 years). The CAD group had a significantly higher level of ox-LDL autoantibody than the non-CAD group (494.0 +/- 355.0 mU/ml vs 258.1 +/- 196.8 mU/ml, p = 0.004). However, the other lipid profiles including triglyceride, total cholesterol, LDL-cholesterol and HDL-cholesterol were not statistically different between the two groups. Forty-six patients in this study had an arterial blood sample taken from the femoral artery for testing ox-LDL autoantibody. There was no significant difference between the arterial and venous samples of ox-LDL autoantibody (385.2 +/- 333.3 mU/ml vs 399.3 +/- 339.5 mU/ml, n = 46, p = 0.530). CONCLUSIONS: Ox-LDL autoantibody was significantly higher in the CAD group. Ox-LDL may prove to play a key role in the pathogenesis of atherosclerosis. Further study of Ox-LDL and its role in the process of atherosclerosis is warranted.

Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs.

Neuropeptide FF (NPFF) has been reported to be an endogenous anti-opioid peptide that has significant effects on morphine tolerance and dependence. In the present study, we examined the chronic effects of NPFF and its synthetic analogs: the putative agonist, PFRFamide, and the putative antagonists, dansyl-PQRamide and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes in rats. After a 5-day co-administration with morphine [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.)] and the tested peptide [intracerebroventricularly (i.c.v.) or i.p., b.i.d.], naloxone (4 mg/kg, i.p.) was given systemically to evaluate the severity of the morphine withdrawal syndromes. Our results revealed that NPFF significantly potentiated the overall morphine withdrawal syndromes and, on the contrary, dansyl-PQRamide attenuated these syndromes. These results clearly indicate that modulation of the NPFF system in the mammalian central nervous system has significant effects on opiate dependence. In addition, morphine withdrawal syndromes could be practically applied as a valid parameter to functionally characterize the putative NPFF agonists and antagonists.

Unilateral pulmonary artery agenesis with presentation of hemoptysis: a case report.

Unilateral pulmonary artery agenesis (UPAA), a rare congenital anomaly frequently associated with other cardiovascular abnormalities, is usually diagnosed and surgically treated in childhood. Those who do not suffer other cardiac anomalies (isolated UPAA) have only minor or no symptoms and survive into adulthood. Isolated UPAA in adult patients may present as recurrent respiratory tract infection, dyspnea on exertion, hemoptysis or an incidental finding of an abnormal chest radiograph. We present the case of a 38-year-old man with a congenital absence of the right pulmonary artery (PA) and recurrent hemoptysis. The diagnosis was confirmed by cardiac catheterization, which disclosed an absence of the right PA and systemic collaterals to the right lung from the right internal thoracic artery and posterior intercostal arteries.

Retrovirus-mediated transfer of anti-MDR1 ribozymes fully restores chemosensitivity of P-glycoprotein-expressing human lymphoma cells.

Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-MDR1 hammerhead ribozymes (Rzs), one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second a stem II base-modified (U9-->Gg, U13-->A13, G14-->A14, A18-->C18) Rz against codon 196 (196MDR1-sRz), and the third a stem II base-modified Rz directed against the -6 approximately -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N2A+tRNAiMet downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A+tRNAiMet-196MDR1-Rz, N2A+tRNAiMet-196MDR1-sRz, and N2A+tRNAiMet-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retroviral vectors. The supernatants containing the packaged retrovirus in high titers (1.1-2.5 X 10(5) CFU/ml as determined by infection of NIH 3T3 cells) were used to infect Daudi/MDR20 cells. The iMDR1-sRz- and 196MDR1-sRz-transduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and doxorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-glycoprotein as well as overexpression of anti-MDR1 Rz. In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activities than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identified to enhance the cleavage efficacy. The combination of these two factors, together with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.

Morphine tolerance in arthritic rats and serotonergic system.

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.

Neuropeptide FF potentiates the behavioral sensitization to amphetamine and alters the levels of neurotransmitters in the medial prefrontal cortex.

We have demonstrated that chronic administration of neuropeptide FF (NPFF) into the lateral ventricle potentiated the behavioral sensitization to amphetamine. Further, the treatment with NPFF decreased the levels of serotonin, and increased the glutamate and GABA content in the medial prefrontal cortex of amphetamine-sensitized rats. The results suggest that NPFF may modulate the neuronal process of amphetamine addiction.