3-Deazaadenosine mitigates arterial remodeling and hypertension in hyperhomocysteinemic mice

Chronic hyperhomocysteinemia (HHcy) is an important factor in development of arterial hypertension. HHcy is associated with activation of matrix metalloproteinases (MMPs); however, it is unclear whether HHcy-dependent extracellular matrix (ECM) accumulation plays a role in arterial hypertrophy and hypertension. We tested the hypothesis that in HHcy the mechanism of arterial hypertension involves arterial dysfunction in response to ECM accumulation between endothelial and arterial smooth muscle cells and subsequent endothelium-myocyte (E-M) uncoupling. To decrease plasma Hcy, dietary supplementation with 3-deazaadenosine (DZA), the S-adenosylhomocysteine hydrolase inhibitor, was administered to cystathionine beta-synthase (CBS) knockout (KO) mice. Mice were grouped as follows: wild type (WT; control), WT+DZA, CBSKO, and CBSKO+DZA (n = 4/group). Mean aortic blood pressure and heart rate were monitored in real time with a telemetric system before, during, and after DZA treatment (6 wk total). In vivo aorta function and morphology were analyzed by M-mode and Doppler echocardiography in anesthetized mice. Aorta MMP activity in unfixed cryostat sections was measured with DQ gelatin. Aorta MMP-2, MMP-9, and connexin 43 expression were measured by RT-PCR and Western blot analyses, respectively. HHcy caused increased aortic blood pressure and resistance, tachycardia, and increased wall thickness and ECM accumulation in aortic wall vs. control groups. There was a linear correlation between aortic wall thickness and plasma Hcy levels. MMP-2, MMP-9, and connexin 43 expression were increased in HHcy. In the CBSKO+DZA group, aortic blood pressure and levels of MMP and connexin 43 were close to those found in control groups. However, removal of DZA reversed the aortic lumen-to-wall thickness ratio in CBSKO mice, suggesting, in part, a role of vascular remodeling in the increase in blood pressure in HHcy. The results show that arterial hypertension in HHcy mice is, in part, associated with arterial remodeling and E-M uncoupling in response to MMP activation.     

The Water Channel Aquaporin 1 Is a Novel Molecular Target of Polychlorinated Biphenyls for in Utero Anomalies

Despite serious health risks in humans and wild life, the underlying mechanisms that explain the gene-environment effects of chemical toxicants are largely unknown. Polychlorinated biphenyls (PCBs) are one of the most ubiquitous environmental toxicants worldwide, with reported epidemiological evidence for reproductive and neurocognitive anomalies in humans. Here, we show that Aroclor 1254, a mixture of structurally distinct PCBs, causes preterm birth in interleukin (IL)-10^-/- mice at a dose that does not show any adverse effects in wild type mice, highlighting the significance of IL-10 as an anti-toxicant cytokine. Aroclor 1254-treated IL-10^-/- mice demonstrated increased amniotic fluid, intrauterine growth restriction, and reduced litter size with postnatal neuromotor defects. Further, our results identify aquaporin 1 (AQP1), a potent effector of fluid volume regulation and angiogenic activity, as a novel placental target of PCBs. In vivo or in vitro exposure to Aroclor 1254 coupled with IL-10 deficiency significantly reduced the protein content of AQP1. Reduced uterine AQP1 levels were associated with defective spiral artery transformation. Importantly, recombinant IL-10 reversed PCB-induced in vivo and in vitro effects. These data demonstrate for the first time that the IL-10-AQP1 axis is a novel regulator of PCB-induced in utero effects.The health consequences of environmental toxicants are likely to have critical effects during in utero fetal development because of the complex signaling cascades, high cellular proliferation rates, and differentiation events. Mammalian reproduction involves a complex but highly choreographed sequence of molecular processes. These processes include interactions between the hormonally stimulated uterus and the developing blastocyst, implantation, placental and fetal development, and parturition (1, 2). Although the hormonal milieu, metabolic changes, and placental microenvironment are programmed in a pregnancy compatible manner, pregnancy presents itself as an immunological and hormonal paradox (3, 4). The role of steroid hormones is well known in uterine receptivity, implantation, local immune modulation, and pregnancy success (5). If not temporally produced and regulated, their dysfunction lead to infertility or pregnancy loss. Man-made chemicals like polychlorinated biphenyls (PCBs)² act like hormones and interfere with their cognate receptor functions impacting normal biological processes (6, 7). Although the genotoxic effects of PCBs have been investigated intensively and epidemiological studies have highlighted their health risks (6, 7), the mechanisms responsible for reproductive and neurodevelopmental effects still remain enigmatic. The overarching goal of our studies is to identify unknown pathways and targets that impart adverse effects on pregnancy. In this study, we directed our efforts toward establishing an experimental system to evaluate the in utero gene-environment effects of PCBs using wild type mice and their counterparts deficient in pregnancy compatible anti-inflammatory cytokines such as interleukin 10 (IL-10).IL-10 is a potent anti-inflammatory cytokine that controls inflammatory insult in most organs, particularly at the maternal-fetal interface. IL-10 is produced by gestational tissue and maternal immune cells in the intrauterine microenvironment in humans (8, 9) and in mice (10). We and others have reported that IL-10^-/- mice experience preterm birth and resorptions in response to low doses of inflammatory triggers such as lipopolysaccharide (LPS) (11, 12) or poly(I-C) (13). Importantly, the pregnancy outcome in treated IL-10^-/- mice can be rescued by giving an exogenous dose of IL-10 (11, 14). We have also demonstrated poor IL-10 production in placental and decidual tissues from preterm labor deliveries and missed abortions (15, 16). These data suggest that an inflammatory environment coupled with genetic stress (IL-10 deficiency) may lead to adverse pregnancy outcomes. In consideration of these observations, we hypothesize that exposure to toxicants such as PCBs mimics the physiological counterpart of inflammation that predisposes to adverse pregnancy outcomes when combined with genetic deficiency in loci crucial for pregnancy success such as IL-10.PCBs are chlorinated aromatic hydrocarbon compounds consisting of a group of 209 structurally diverse congeners, identified based on the position of chlorine atoms (7). Since the start of their manufacture in the 1920s until their ban in late 1970s, PCBs were globally valued for their noninflammability and high heat and chemical stability and thus were used widely in a multitude of commercial and industrial applications (7, 17). Improper disposal and accidental release of these compounds led to their introduction into the environment, placing them in the list of widespread environmental contaminants. Subsequently, their lipophilicity facilitated their bioaccumulation in the food chain and bio-concentration at successively higher levels (6, 18-21). PCBs have now been detected globally, in different environmental matrices, wild life, food, and humans (6, 18, 20). Convincing evidence exist for their toxicity, both in humans as well as in laboratory animals (7). From epidemiological studies in humans it has been observed that exposure to PCBs causes various reproductive anomalies that include irregular and shorter menstrual cycles, delayed conception, miscarriage, reduced lactating time, low birth weight, preterm birth, small for gestational age infants, and higher incidence of still-births and mortality among children (22-27). PCB congeners may work in an aryl hydrocarbon receptor-dependent or -independent pathway (6, 7, 28). Despite the knowledge that PCBs affect either aryl hydrocarbon receptor or estrogen receptor signaling, there is a paucity of molecular mechanisms underlying the most sensitive developmental effects of PCBs, and thus new pathways and targets need to be identified.Aroclor 1254 is a mixture of more than one hundred different PCB congeners and may impart cumulative adverse effects on female reproductive health (29, 30). In this study, we show that Aroclor 1254 exposure induces preterm birth in IL-10^-/- mice with reduced litter size and birth weight, increased amniotic fluid, and postnatal neurocognitive defects. Importantly, we have identified aquaporin 1 (AQP1) as a novel target of PCB action at the maternal-fetal interface. Our findings for the first time provide direct experimental evidence for a protective role of IL-10 against PCB exposure. These findings may have implications for the understanding and management of environmental toxicant-induced female reproductive anomalies in humans.     

Spatially variable natural selection and the divergence between parapatric subspecies of lodgepole pine (Pinus contorta, Pinaceae)

? Premise of the study: Plant populations arrayed across sharp environmental gradients are ideal systems for identifying the genetic basis of ecologically relevant phenotypes. A series of five uplifted marine terraces along the northern coast of California represents one such system where morphologically distinct populations of lodgepole pine (Pinus contorta) are distributed across sharp soil gradients ranging from fertile soils near the coast to podzolic soils ca. 5 km inland. ? Methods: A total of 92 trees was sampled across four coastal marine terraces (N = 10-46 trees/terrace) located in Mendocino County, California and sequenced for a set of 24 candidate genes for growth and responses to various soil chemistry variables. Statistical analyses relying on patterns of nucleotide diversity were employed to identify genes whose diversity patterns were inconsistent with three null models. ? Key results: Most genes displayed patterns of nucleotide diversity that were consistent with null models (N = 19) or with the presence of paralogs (N = 3). Two genes, however, were exceptional: an aluminum responsive ABC-transporter with F(ST) = 0.664 and an inorganic phosphate transporter characterized by divergent haplotypes segregating at intermediate frequencies in most populations. ? Conclusions: Spatially variable natural selection along gradients of aluminum and phosphate ion concentrations likely accounted for both outliers. These results shed light on some of the genetic components comprising the extended phenotype of this ecosystem, as well as highlight ecotones as fruitful study systems for the detection of adaptive genetic variants.     

Nocturnal energy demand in plants: Insights from studying mutants impaired in β-oxidation

All photosynthetic organisms face the difficulty of maintaining cellular metabolism in the absence of photosynthetic active radiation during the night. Although many consuming metabolic pathways (e.g., fatty acid synthesis) are only active in the light, plant cells still require basic levels of metabolic energy and reductive power during the night for sustained growth and development.Key words: PXA1, comatose, β-oxidation, fatty acids, starch, imaging PAM, extended darkness     

16S rRNA gene profiling of rhizospheric microbial community of Eichhornia crassipes

Molecular Biology Reports - The rhizosphere of a plant is an important interface for the plant-microbe interaction that plays a significant role in the uptake and removal of heavy metal from...     

Interaction of dihydrofolate reductase and aminoglycoside adenyltransferase enzyme from Klebsiella pneumoniae multidrug resistant strain DF12SA with clindamycin: a molecular modelling and docking study

Klebsiella pneumoniae strain DF12SA (HQ114261) was isolated from diabetic foot wounds. The strain showed resistance against ampicillin, kanamycin, gentamicin, streptomycin, spectinomycin, trimethoprim, tetracycline, meropenem, amikacin, piperacillin/tazobactam, augmentin, co-trimoxazole, carbapenems, penicillins and cefoperazone, and was sensitive to clindamycin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing two genes, a dihydrofolate reductase (DHFR) (PF00186), which confers resistance to trimethoprim; and aminoglycoside adenyltransferase (AadA) (PF01909), which confers resistance to streptomycin and spectinomycin. A class 1 integron in K. pneumoniae containing these two genes was present in eight (18.18 %) out of 44 different diabetic foot ulcer (DFU) patients. Hence, there is a need to develop therapeutics that inhibit growth of multidrug resistant K. pneumoniae in DFU patients and still achieve amputation control. Am attempt was made to create a 3D model and find a suitable inhibitor using an in silico study. Rational drug design/testing requires crystal structures for DHFR and AadA. However, the structures of DHFR and AadA from K. pneumoniae are not available. Modelling was performed using Swiss Model Server and Discovery Studio 3.1. The PDBSum server was used to check stereo chemical properties using Ramachandran plot analysis of modeled structures. Clindamycin was found to be suitable inhibitor of DHFR and AadA. A DockingServer based on Autodock & Mopac was used for docking calculations. The amino acid residues Ser³², Ile⁴⁶, Glu⁵³, Gln⁵⁴, Phe⁵⁷, Thr⁷², Met⁷⁶, Val⁷⁸, Leu⁷⁹, Ser¹²², Tyr¹²⁸, Ile¹⁵¹ in case of DHFR and Phe³⁴, Asp⁶⁰, Arg⁶³, Gln⁶⁴, Leu⁶⁸, Glu⁸⁷, Thr⁸⁹, Val⁹⁰ for AadA were found to be responsible for positioning clindamycin into the active site. The study identifies amino acid residues crucial to ‘DHFR and AadA -drug’ and ‘DHFR and AadA -inhibitor’ interactions that might be useful in the ongoing search for a versatile DHFR and AadA -inhibitor.     

Synergy of Homocysteine, MicroRNA, and Epigenetics: A Novel Therapeutic Approach for Stroke

Homocysteine (Hcy) is a thiol-containing amino acid formed during methionine metabolism. Elevated level of Hcy is known as hyperhomocysteinemia (HHcy). HHcy is an independent risk factor for cerebrovascular diseases such as stroke, dementia, Alzheimer’s disease, etc. Stroke, which is caused by interruption of blood supply to the brain, is one of the leading causes of death and disability in a number of people worldwide. The HHcy causes an increased carotid artery plaque that may lead to ischemic stroke but the mechanism is currently not well understood. Though mutations or polymorphisms in the key genes of Hcy metabolism pathway have been well elucidated in stroke, emerging evidences suggested epigenetic mechanisms equally play an important role in stroke development such as DNA methylation, chromatin remodeling, RNA editing, noncoding RNAs (ncRNAs), and microRNAs (miRNAs). However, there is no review available yet that describes the role of genetics and epigenetics during HHcy in stroke. The current review highlights the role of genetics and epigenetics in stroke during HHcy and the role of epigenetics in its therapeutics. The review also highlights possible epigenetic mechanisms, potential therapeutic molecules, putative challenges, and approaches to deal with stroke during HHcy.     

Effects of sphingosine-1-phosphate on pacemaker activity of interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P_1,3,4,5 agonist) and SEW 2871 (an S1P₁ agonist) had no effects on pacemaker activity. Suramin (an S1P₃ antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P₂ antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P₂ in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH₂-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca²⁺ ([Ca²⁺]_i) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca²⁺]_i oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P₂ via regulation of external and internal Ca²⁺ and mitogenactivated protein kinase activation.     

Electrical stimulation of cardiomyocytes activates mitochondrial matrix metalloproteinase causing electrical remodeling

Cardiac arrhythmias, instigated by mechanical and electrical remodeling, are associated with activation of extracellular matrix metalloproteinases (MMPs). However, the connection between intracellular MMPs activation and arrhythmogenesis is not well established. Previously, we determined localization of MMP in the mitochondria using confocal microscopy. We tested the hypothesis that electrical pacing induces the activation of mitochondrial MMP (mtMMP) and is associated with myocyte mechanical dysfunction. Myocytes were isolated and field stimulated at 1 and 4 Hz. Myocyte mechanics and calcium transient was studied using Ion-Optix system. Mitochondrial MMP-9 activation was evaluated using zymography. There was a 25% increase in 1 Hz and 40% increase in 4 Hz stimulation. We observed an increase in mtMMP activation with increase in electrical pacing compared to 0 Hz with a significant increase (p < 0.05, n = 3). Field stimulation at 4 Hz decreased cell re-lengthening. The levels of calcium transient were reduced with increase in contraction frequency. We conclude that electrical stimulation activates mtMMP-9 that is associated with myocyte mechanical dysfunction.