PARP mediates structural alterations in diabetic cardiomyopathy.

Diabetic cardiomyopathy is characterized by structural alterations such as cardiomyocyte hypertrophy, necrosis and focal fibrosis. Hyperglycemia-induced oxidative damage may play an important role in this pathogenetic process. Recent studies have shown that poly (ADP-ribose) polymerase (PARP) is activated in response to oxidative stress and cellular damage as well, plays a role in gene expression. This study investigated mechanisms of diabetes-induced, PARP-mediated development of structural alterations in the heart. Two models of diabetic complications were used to determine the role of PARP in oxidative stress, cardiac hypertrophy and fibrosis in the heart. PARP-1 knockout (PARP(-/-)) mice and their respective controls were fed a 30% galactose diet while male Sprague-Dawley rats were injected with streptozotocin and subsequently treated with PARP inhibitor 3-aminobenzamide (ABA). The in vivo experiments were verified in in vitro models which utilized both neonatal cardiomyocytes and endothelial cells. Our results indicate that hyperhexosemia caused upregulation of extracellular matrix proteins in association with increased transcriptional co-activator p300 levels, cardiomyocyte hypertrophy and increased oxidative stress. These pathogenetic changes were not observed in the PARP(-/-) mice and diabetic rats treated with ABA. Furthermore, these changes appear to be influenced by histone deacetylases. Similar results were obtained in isolated cardiomyocytes and endothelial cells. This study has elucidated for the first time a PARP-dependent, p300-associated pathway mediating the development of structural alterations in the diabetic heart.

Oxidative stress-induced, poly(ADP-ribose) polymerase-dependent upregulation of ET-1 expression in chronic diabetic complications.

Hyperglycemia in diabetes induces increased endothelin-1 (ET-1) production in the retina, kidney, and heart that may lead to hemodynamic impairment, permeability alteration, and increased extracellular matrix (ECM) protein production. Chronically elevated blood glucose levels may cause oxidative stress in these target tissues of diabetic complications. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks due to oxidative stress. We investigated the role of PARP in regulating ET-1 expression and ET-1-induced abnormalities in the targets organs of diabetic complications. Male Sprague-Dawley rats were injected with streptozotocin to induce diabetes. Once diabetes was established, half of the diabetic rats were randomly chosen to receive PARP inhibitor 3-aminobenzamide for 4 months. In a second set of experiments, PARP-/- mice and their controls were fed for 2 months with either a normal rodent diet or a 30% galactose diet to induce a normoinsulinemic hyperhexosemic state. Tissues harvested at the conclusion of both experiments were then subjected to real-time RT-PCR analysis for mRNA expression and immunohistochemical assessment of oxidative stress. In both experiments, the hyperhexosemic state upregulated expression of ET-1 mRNA in the retina, kidney, and heart. Furthermore, upregulation of ET-1-dependent ECM transcripts, such as fibronectin and extradomain B-containing fibronectin, was noted in all tissues. These tissues also demonstrated oxidative stress, as evidenced by the presence of nuclei positive for 8-hydroxy-2'-deoxyguanosine. In contrast, inhibition of PARP, either through a chemical means in the diabetic rats or by genetic manipulation in the galactose-fed animals, prevented both oxidative stress and hyperhexosemia-induced upregulation of these genes. These results suggest that, in diabetes, oxidative stress and PARP activation may produce their effects through ET-1. Hence, blockade of such pathways may constitute potential adjuvant treatment modalities in chronic diabetic complications.

[Extraction and analysis of nuclear DNA from free margin of nail material].

To investigate the feasibility of DNA analysis from free margin of the nail, genomic DNA was extracted from the free margin of nail clipping of 10 volunteers using the proteinase K/SDS -based organic method, the Chelex-100 method, or a combined method. Target DNA was simultaneously amplified using a fluorescent multiplex AmpFlSTR Identifier kit. The PCR products were analyzed on the ABI PRISM 3130 Genetic Analyzer. The results showed that, compared with profiles achieved by genotyping of blood samples from each volunteer as reference, 100% concordance was achieved using the combined method. The STR genotype profiles obtained through the organic method were acceptable, despite preferential amplification at some loci. In contrast, no readable profiles could be determined when DNA was extracted by the Chelex-100 method, and there were a large number of alleles missing. Our data suggest that free margin of nail can be used for nuclear DNA analysis, but the type of DNA isolation method used is critical. The traditional organic extraction method works reasonably well for free margin nail DNA isolation, and combination of organic extraction and the Chelex-100 method works best.

[Variation of BDNF mRNA on focalcerebral ischemia reperfusion injury in rats with notogisenoside-Rg1].

OBJECTIVE: To study the effect of notoginsenoside-Rg, on expression of BDNF mRNA (brain-derived neurotrophic factor messenger ribonucleic acid) in cerebrum cortex after MCAO/R (middle cerebral artery occlusion reperfusion) injury in rat by real-time quantitative polymerase chain reaction. METHODS: 36 SD male rats were randomly divided into MCAO/R model group, notogisenoside-Rg1 therapy group (100 mg/kg) and the positive medicine control group (nimodipine 1 mg/kg). The MCAO/R model were made by thread-occluded method. The four rats were randomly taken from each groups and were killed to be breaken out brain tissues as specimens after which were treated with medicine in 1,3, 5 days. Total RNA was isolated from cerebrum cortex. Specific oligonucleotide primers of BDNF mRNA gene fragments were amplificated by RT-PCR to construct recombinant plasmid and sequence. To dilute recombinant plasmid didploidly and a quantitative standard curve was completed. Taqman fluorogenic quantitative RT-PCR (FQ-PCR) was set up to detect the BDNF mRNA variety of cerebrum cortex. RESULTS: Compared with the model group and the postive control group, notogisenoside-Rg1 treated groups could obviously improve some nervous deficit symptoms in the cerebral ischemia and increase BDNF mRNA amount that in the cerebrum cortex at different period after rat MCAO/R injury. CONCLUSION: Notoginsenoside-Rg1 can promote to generating internal BDNF protein in brain by up-regulation the expression of BDNF mRNA amount in the cerebrum cortex. BDNF bind in TrkB, which can give rise to protective effects for ischemic neurons by generating corresponding domino effect molecules. Accordingly it can be as a therapy method in cerebral ischemia injury.

Genetic variability of CYP2B6 polymorphisms in four southern Chinese populations.

AIM: To investigate the genotype and allelic frequencies of Cytochrome P450 2B6 polymorphisms in four southern Chinese populations. METHODS: DNA was obtained from blood samples from Han Chinese from Hong Kong and three minority groups, the Wa, Bulang and Lahu from Yunnan in southern China. Genotyping was performed using real-time PCR and confirmed by direct sequencing. RESULTS: A total of 507 subjects from southern China were studied. Results showed there is a high prevalence of 516G > T (34.5%) in ethnic Chinese compared to literature reports on other Asian populations and Caucasians. The frequency of the 516TT genotype is higher in the Han majority (23.1%) than in three other ethnic minority groups (i.e., 7.4%, 9.1% and 15.8%) in southern China. CONCLUSION: This was the first study to document the spectrum of CYP2B6 allelic variants and genotypes in a southern Chinese population. The 516G > T allele is associated with a defective metabolism of efavirenz (EFV), which therefore may predispose to drug toxicity. Treatment regimens for human immunodeficiency virus (HIV) and heroin addiction may need to be optimized in different populations because of the marked variability of the key metabolizing enzyme.

[Comparison of retrograde amnesia changes within different injury levels of cerebral concussion in rats].

OBJECTIVE: To investigate the retrograde amnesia changes within different injury levels of cerebral concussion in rats. METHODS: A metallic pendulum striker device of brain injury was deployed to duplicate CC models of different injury levels within Sprague-Dawley (S-D) rats. The investigated animals were divided into two groups according to classification standard, that is, Pure Cerebral Concussion (PCC) group and Complicated Cerebral Concussion (CCC) group. One control group was used, and each group included 8 animals. The retrograde amnesia of each group was assessed by Morris Water Maze (MWM) Test from 3 days preinjury to 7 days postconcussion. RESULTS: Compared with the control group, the retrograde amnesia was detected within 3 days in PCC group, and 5 days in CCC group after injury. At the same time, the two groups both manifested space recognition deficit. CONCLUSION: The retrograde amnesia existed in both pure cerebral concussion group and complicated cerebral concussion. Furthermore, the lasting time of retrograde amnesia in animals correlates to the injury level of brain concussion.