Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively. Levels of 8-OHdG and AP sites were markedly elevated 16-72 h following MCAO in the frontal cortex, representing the peri-infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT- and TUNEL-positive cells began to be detectable 4-8 h following MCAO in the caudate-putamen and parietal cortex and reached maximal levels at 72 h. PANT- and TUNEL-positive cells were also detected 16-72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single-strand breaks and DNA fragmentation. In contrast, levels of PANT-positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri-infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.     

Risk factors of peripheral arterial disease: a case control study in Sri Lanka

Background

Peripheral artery disease (PAD) is an important global health problem and contributes to notable proportion of morbidity and mortality. This particular manifestation of systemic atherosclerosis is largely under diagnosed and undertreated. For sustainable preventive strategies in a country, it is mandatory to identify country-specific risk factors. We intended to assess the risk factors of PAD among adults aged 40–74 years.

Methods

This case control study was conducted in 2012–2013 in Sri Lanka. Seventy-nine cases and 158 controls in the age group of 40–74 years were selected for the study in order to have case to control ratio 1:2. The criterion for selecting cases and control was based on Ankle brachial pressure index (ABPI). Cases were selected from those who had ABPI 0.85 or less (ABPI ≤0.85) in either lower limb. Controls were selected from those ABPI score between 1.18 and 1.28 in both lower limbs. Only newly identified individuals with PAD were selected as cases. Controls were selected from the same geographical location and within the 5 year age group as cases.

Results

The history of diabetes mellitus more than 10 years (OR 5.8, 95% CI 2.2–14.2), history of dyslipidemia for more than 10 years (OR 4.9, 95% CI 2.1–16.2), history of hypertension for more than 10 years (OR 3.8, 95% CI 1.8–12.7) and smoking (OR 2.9, 95% CI 1.2–6.9), elevated HsCRP (OR 3.7, 95% CI 1.2–12.0) and hyperhomocysteinemia (OR 3.0, 95% CI 1.1–8.1) were revealed as country specific significant risk factor of PAD.

Conclusions

Diabetes mellitus, hypertension, dyslipidemia, smoking as well as elevated homocysteine and HsCRP found as risk factors of PAD. Longer the duration or higher level exposure to these risk factors has increased the risk of PAD. These findings emphasis the need for routine screening of PAD among patients with the identified risk factors.

     

Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures

Death-associated protein (DAP) kinase is calcium-regulated and known to function downstream of death receptors, prompting us to examine its role in the mechanism of seizure-induced neuronal death. Brief seizures were focally evoked in rats, eliciting neuronal death within the CA3 subfield of the hippocampus, and to a lesser extent, cortex. Western blotting confirmed expression of DAP kinase within hippocampus and cortex at the predicted weight of approximately 160 kDa. Immunohistochemistry revealed seizures triggered a significant increase in numbers of DAP kinase-expressing cells within CA3 and cortex, without affecting cell counts within seizure-resistant CA2 or the dentate gyrus. Numbers of DAP kinase-expressing cells were increased in relation to specific patterns of injury-causing seizure activity, electrographically defined. Seizures caused an early increase in DAP kinase binding to actin, and association with calmodulin. Co-immunoprecipitation studies also revealed seizures triggered binding of DAP kinase to the tumor necrosis factor receptor 1 and the Fas-associated death domain protein, commensurate with caspase-8 proteolysis. In contrast, within surviving fields of the hippocampus, DAP kinase interacted with the molecular chaperone 14-3-3. These data suggest DAP kinase is involved in the molecular pathways activated during seizure-induced neuronal death.     

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.     

Factors influencing the efficiency of a marker-assisted introgression programme in Merino sheep

This study investigated a marker-assisted introgression programme in Australian Merino sheep. The goal was to introgress an allele with a large negative effect on fibre diameter into a Merino flock possessing medium average fibre diameter. The influence of two factors was explored: the strategy used to select animals from the purebred and backcross line for backcrossing purposes and the use of selection on background markers to accelerate the return to the purebred line''s genome. The results were compared to introgression based on EBVs only. Introgression using EBVs only produced almost the same response in the dollar index as marker-based introgression methods. However, this study did not account for some of the costs associated with implementing the programmes, including the costs of phenotyping and genotyping. Given that the cost of measuring fibre diameter is low, it was concluded that introgression on EBVs only would be the preferred method since the marginal profit of marker-assisted introgression would not be large enough to cover the additional cost of genotyping. In marker-assisted introgression, reciprocal crossing of male and female selection candidates from the backcross and the purebred line was the most advantageous strategy from a practical and profit point of view. Selection for background markers was less profitable in this study than recovering the donor genome by selection on phenotype.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Mutation of Semaphorin-6A disrupts limbic and cortical connectivity and models neurodevelopmental psychopathology

Psychiatric disorders such as schizophrenia and autism are characterised by cellular disorganisation and dysconnectivity across the brain and can be caused by mutations in genes that control neurodevelopmental processes. To examine how neurodevelopmental defects can affect brain function and behaviour, we have comprehensively investigated the consequences of mutation of one such gene, Semaphorin-6A, on cellular organisation, axonal projection patterns, behaviour and physiology in mice. These analyses reveal a spectrum of widespread but subtle anatomical defects in Sema6A mutants, notably in limbic and cortical cellular organisation, lamination and connectivity. These mutants display concomitant alterations in the electroencephalogram and hyper-exploratory behaviour, which are characteristic of models of psychosis and reversible by the antipsychotic clozapine. They also show altered social interaction and deficits in object recognition and working memory. Mice with mutations in Sema6A or the interacting genes may thus represent a highly informative model for how neurodevelopmental defects can lead to anatomical dysconnectivity, resulting, either directly or through reactive mechanisms, in dysfunction at the level of neuronal networks with associated behavioural phenotypes of relevance to psychiatric disorders. The biological data presented here also make these genes plausible candidates to explain human linkage findings for schizophrenia and autism.     

Assessment of pharmacological activities of two medicinal plant of Bangladesh: Launaea sarmentosa and Aegialitis rotundifolia roxb in the management of pain,pyrexia and inflammation

Background

The current study aims at evaluating the analgesic, anti-pyretic and anti-inflammatory properties of methanolic extract of the stem, bark and leaves of Launaea sarmentosa and Aegialitis rotundifolia roxb.

Results

The AELS and AEAR extract presented a significant (***p < 0.001) dose dependent increase in reaction time in writhing method and showed inhibition of 63.1% and 57.1% respectively at the doses of 400 mg/kg body weight while standard drug showed (P < 0.001) inhibition of 69.23%. In tail immersion method, AELS and AEAR showed maximum time of tail retention at 30 min in hot water i.e. 6.93 sec and 6.54 sec respectively at highest doses of 400 mg/kg body weight than lower dose while standard pentazocine showed reaction time of 7.62 sec. The AELS and AEAR extract also exhibited promising anti-inflammatory effect as demonstrated by statistically significant inhibition of paw volume by 32.48% and 26.75% respectively at the dose of 400 mg/kg body weight while the value at the dose of 200 mg/kg body weight were linear to higher dose at the 3^rd hour of study. On the other hand, Standard indomethacin inhibited 40.13% of inflammation (***P < 0.001). In Cotton-pellet granuloma method, AELS and AEAR extract at the dose of 400 mg/kg body weight exhibited inhibition of inflammation of 34.7% and 29.1% respectively while standard drug showed (P < 0.001) inhibition of 63.22%. Intraperitoneal administration of AELS and AEAR showed dose dependent decrease in body temperature in brewer’s yeast induced hyperthermia in rats at both doses. However, AELS significantly decreased body temperature (***p < 0.001) at 400 mg/kg compared to control.

Conclusions

Present work propose that the methanolic extract of Launaea sarmentosa and Aegialitis rotundifolia roxb possesses dose dependent pharmacological action which supports its therapeutic use in folk medicine possibly mediated through the inhibition or blocking of release of prostaglandin and/or actions of vasoactive substances such as histamine, serotonin and kinins.