Oligomeric proanthocyanidins protect myocardium by mitigating left ventricular remodeling in isoproterenol‐induced postmyocardial infarction

Extracellular matrix (ECM) remodeling is a major pathophysiological process during post‐myocardial infarction (MI). The activation, differentiation, and proliferation of cardiac fibroblasts to myofibroblasts regulate the expression of ECM proteins. The signaling by bone morphogenetic protein (BMP‐4), an extracellular ligand of the TGF‐β family, has recently been identified as an essential pathway in regulating cardiovascular dysfunctions including myocardial fibrosis. Oligomeric proanthocyanidins (OPC) are well known for their cardioprotective activity. The primary aim of the study was to investigate BMP‐4‐mediated ECM turnover in cardiac fibrosis during isoproterenol‐induced post‐MI and its downregulation by OPC. Myocardial injury was evaluated by assaying serum markers LDH and CK. Oxidative stress and the enzymatic and nonenzymatic antioxidant levels were assessed to support the cardioprotective nature of OPC. The total collagen level was analyzed by measuring hydroxyproline levels. The ISO‐induced group showed a significant decrease in the levels of antioxidants due to severe oxidative stress and increased expression of BMP‐4 which reflects the increased expression of MMP 2 and 9 with a concomitant increase and deposition of fibrillary collagens type I and III responsible for the fibrotic scar formation as evidenced in the histological analysis.BMP‐4 activation, thus, is strongly associated with cardiac fibrosis which was downregulated upon OPC supplementation. This study provides an evidence supporting the antifibrotic effect of OPC via regulation of BMP‐4‐mediated ECM turnover and also substantiates the remarkable antioxidant efficacy of OPC against isoproterenol induced severe oxidative stress and subsequent post‐MI cardiac fibrosis.     

Antiulcerogenic activity of hydroalcoholic fruit extract of Pithecellobium dulce in different experimental ulcer models in rats

Ethnopharmacological relevance

The ethnopharmacological importance of Pithecellobium dulce is evidenced by its traditional use for gastric complications. The aim of the study is to evaluate the gastroprotective activity and the mechanism of action of hydroalcoholic fruit extract of P. dulce (HAEPD) in rats by using chemical and stress induced ulcer models.

Materials and methods

Gastric ulcer was induced by administering alcohol (or) acetylsalicylic acid (or) hypothermic restraint stress to rats pretreated with HAEPD (200 mg/kg b wt for 30 day). Volume of gastric fluid, pH, acidity, activities of pepsin, H⁺, K⁺-ATPase, myeloperoxidase, mucin content, nucleic acids, glycoproteins and prostaglandin E₂ (PGE₂) levels were assessed in gastric tissues.

Results

Ulcer score was significantly minimized in HAEPD administered animals. pH and acidity of gastric fluid were significantly minimized and the mucin, PGE₂ levels were significantly maintained in drug pre administered animals. The activities of H⁺, K⁺- ATPase and myeloperoxidase were found to be significantly elevated in ulcer control animals and found to be decreased in drug pretreated animals. The cell proliferation was found to be enhanced in drug received animals. The total protein bound carbohydrate to total protein ratio was found to be significantly maintained by HAEPD. The effects were found to be comparable with that of standard drug omeprazole.

Conclusion

It is concluded that HAEPD possess a potent antiulcer activity probably by acting as cytoprotective and antiacid secretory agent.     

The Na–K–Cl Co-transporter in astrocyte swelling

Ion channels, exchangers and transporters are known to be involved in cell volume regulation. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na⁺–K⁺–Cl⁻ cotransporters has been shown to regulate cell volume in many conditions. The Na⁺–K⁺–Cl− cotransporters (NKCC) are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. Studies have established the role of NKCC1 in astrocyte swelling/brain edema in ischemia and trauma. Our recent studies suggest that NKCC1 activation is also involved in astrocyte swelling induced by ammonia and in the brain edema in the thioacetamide model of acute liver failure. This review will focus on mechanisms of NKCC1 activation and its contribution to astrocyte swelling/brain edema in neurological disorders, with particular emphasis on ammonia neurotoxicity and acute liver failure.     

NMDA receptors regulate developmental gap junction uncoupling via CREB signaling

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.     

Mutagenesis studies with four stereoisomeric N2-dG benzo[a]pyrene adducts in the identical 5'-CGC sequence used in NMR studies: G->T mutations dominate in each case

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N2-dG in DNA. B[a]P-N2-dG adducts have been shown to induce a variety of mutations, notably G-->T, G-->A, G-->C and -1 frameshifts. Four stereoisomers of B[a]P-N2-dG (designated: [+ta]-;, [+ca]-, [-ta] and [-ca]) were studied by NMR in duplex 11mers in a 5'-CGC sequence context, and each adopted a different adduct conformation (Geacintov, et al. (1997) Chem. Res. Toxicol., 10, 111). Herein these four identical B[a]P-containing 11mers are built into duplex plasmid genomes and mutagenesis studied in Escherichia coli following SOS-induction. In nucleotide excision repair (NER) proficient E.coli, no adduct-derived mutants are detected. In NER deficient E.coli, G-->T mutations dominate for all four stereoisomers [+ta]-, [+ca]-, [-ta] and [-ca]-B[a]P-N(2)-dG, and mutation frequency is similar. Thus, the mutagenic pattern for these four B[a]P-N2-dG stereoisomers is the same, in spite of the fact that they adopt dramatically different conformations in ds-oligonucleotides as determined by NMR. These findings suggest that adduct conformation must be fluid enough in the 5'-CGC sequence that the duplex DNA conformation can interconvert to mutagenic and non-mutagenic conformations during lesion-bypass. A comparison of all published studies with these four B[a]P-N2-dG stereoisomers in E.coli reveals that B[a]P-N2-dG adduct stereochemistry tends to have a lesser impact on mutagenic pattern (e.g. G-->T versus G-->A mutations) than does DNA sequence context, which is discussed.     

Toll-like receptor 2 mediates inflammatory cytokine induction but not sensitization for liver injury by Propioni- bacterium acnes

Recognition of Gram-positive bacteria by Toll-like receptor 2 (TLR2) induces activation of proinflammatory pathways. In mice, sensitization with the Gram-positive Propionibacterium acnes followed by a challenge with the TLR4 ligand, lipopolysaccharide (LPS), results in fulminant hepatic failure. Here, we investigated the role of TLR2 in liver sensitization to LPS-induced injury. Stimulation of Chinese hamster ovary cells and peritoneal macrophages with heat-killed P. acnes required expression of TLR2 but not of TLR4, suggesting that P. acnes was a TLR2 ligand. Cell activation by P. acnes was myeloid differentiation primary-response protein 88 (MyD88)-dependent, and it was augmented by coexpression of CD14 in mouse peritoneal macrophages. In vitro, P. acnes behaved as a TLR2 ligand and induced TLR4 hetero- and TLR2 homotolerance in peritoneal macrophages. In vivo priming of wild-type mice with P. acnes, but not with the selective TLR2 ligands peptidoglycan and lipotheicoic acid, resulted in hepatocyte necrosis, hyperelevated serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, interferon-gamma (IFN-gamma), and IL-12 (p40/p70), and increased RNA expression of proinflammatory cytokines (IL-12p40, IL-1alpha, IL-6, IL-1beta, IL-18, IFN-gamma) in the liver after a LPS challenge. Furthermore, P. acnes priming sensitized TLR2-deficient (TLR2-/-) but not MyD88-/- mice to LPS-induced injury, evidenced by hepatocyte necrosis, increased levels of serum TNF-alpha, IFN-gamma, IL-6, and liver proinflammatory cytokine mRNA expression. IFN-gamma, a cytokine sensitizing to endotoxin, was induced by P. acnes in splenocytes of TLR2-/- and TLR9-/- but not MyD88-/- mice. These results suggest that although P. acnes triggers TLR2-mediated cell activation, TLR2-independent but MyD88-dependent mechanisms mediate in vivo sensitization by P. acnes for LPS-induced liver injury.     

Mutagenesis studies of the major benzo[a]pyrene N2-dG adduct in a 5'-TG versus a 5'-UG sequence: removal of the methyl group causes a modest decrease in the [G->T/G->A] mutational ratio

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations primarily at the G:C base pairs (e.g. GC-->TA, GC-->AT, etc.). Each of these mutations can be induced by its major adduct [+ta]-B[a]P-N(2)-dG, where DNA sequence context appears to influence both the quantitative and qualitative pattern of mutagenesis. We noted previously that 5'-TG sequences tend to have a higher fraction of G-->T mutations for both [+ta]-B[a]P-N(2)-dG and (+)-anti-B[a]PDE in comparison with 5'-CG, 5'-GG or 5'-AG sequences. To investigate a possible structural element for this trend, the role (if any) of the methyl group on the 5'-T is considered. Using adduct site-specific means, the [G-->T/G-->A] mutational ratio for [+ta]-B[a]P-N(2)-dG is determined to be approximately 1.08 in a 5'-TGT sequence, and approximately 0.60 in a 5'-UGT sequence. (G-->C mutations are minor.) Although this modest approximately 1.8-fold decrease in [G-->T/G-->A] ratio is statistically significant (P = 0.03), it suggests that the methyl group on the 5'-T is not the main reason why a 5'-T tends to enhance G-->T mutations. This study was prompted by an adduct conformational hypothesis, which predicted that the removal of the methyl group in a 5'-TG sequence would lower the fraction of G-->T mutations; however, the approximately 1.8-fold decrease is too small to do additional experiments to assess whether this conformational hypothesis, or other hypotheses, are the true cause of the decrease, which is discussed in this paper.     

Mapping of secondary structure of the spacer region within the 5'-untranslated region of the coxsackievirus B3 RNA: possible role of an apical GAGA loop in binding La protein and influencing internal initiation of translation

Translation initiation of the coxsackievirus B3 (CVB3) RNA has been shown to be mediated by a highly ordered structure of the 5'-UTR, which harbors an internal ribosome entry site (IRES). In this study, we have investigated the 48S ribosome assembly site and also characterized the intervening spacer region between the cryptic AUG591 and the initiator AUG742. The ribosomal complex formation was mapped by toe-printing experiment using rabbit reticulocyte lysate, which showed a major toe print at nucleotide U570 corresponding to the 48S ribosome assembly site at the putative Shine-Dalgarno like sequence. Elucidation of the secondary structure of a segment encompassing the ribosome binding site and the downstream spacer region by nuclease probing and chemical modifications demonstrated distinct stem and loop structure. Interestingly, a GAGA loop in the apical region of stem-loop H was found to be phylogenetically conserved as a GNRA-loop among the coxsackie B viruses. Deletion or substitution mutation of this apical GAGA loop drastically reduced binding with human La protein and significantly affected the IRES function. The study revealed important insights into the possible role of the intervening spacer region in cellular protein binding and influencing internal initiation of translation of CVB3 RNA.     

Standardization for image characteristics in telemammography using genetic and nonlinear algorithms

As the soft copy reading and computer assisted diagnosis (CAD) in mammography become more and more important, the standardization of digital images becomes paramount. Telemammography and telemedicine requires the standardization for image characteristics, such as image resolution, bit-depth and intensity response. Soft copy reading and CAD in mammography are both dependent on the characteristics of the source of the digital data, either direct digital mammography or digitized screen-film mammography. An algorithm developed on images from one database may not perform well as on images from another database (with a different digitization). In this paper, we describe two methods based on a genetic algorithm and a nonlinear algorithm for standardization of digitized and digital mammography. The proposed standardization techniques are based on geometric and intensity transformations that are discovered using a set of calibration images. A set of transformation algorithm is used to search for the best standardization.