Monocyclic β-lactam and unexpected oxazinone formation: synthesis,crystal structure,docking studies and antibacterial evaluation

Novel monocyclic β-lactam derivatives bearing aryl, phenyl and heterocyclic rings were synthesized as possible antibacterial agents. Cyclization of imines (3h, 3t) with phenylacetic acid in the presence of phosphoryl chloride and triethyl amine did not afford the expected β-lactams. Instead, highly substituted 1,3-oxazin-4-ones (4h, 4t) were isolated as the only product and confirmed by single crystal X-ray analysis of 4t. The results of antibacterial activity showed that compound 4l exhibited considerable antibacterial activity with MIC and MBC values of 62.5?µg/mL against Klebsiella pneumoniae. Cytotoxicity assay on Chinese Hamster Ovary (CHO) cell line revealed non-cytotoxic behavior of compounds 4d, 4h, 4k and 4l up to 200?μg/mL conc. Molecular docking was performed for compound 4l with penicillin binding protein-5 to identify the nature of interactions. The results of both in silico and in vitro evaluation provide the basis for compound 4l to be carried as a potential lead molecule in the drug discovery pipeline against bacterial infections.     

Nitric Oxide Synthase Expression in Glial Cells: Suppression by Tyrosine Kinase Inhibitors

Abstract: Rat brain glial cells have the capacity to express a calcium-independent form of nitric oxide synthase (iNOS). To test if iNOS induction required tyrosine kinase activity, we made use of genistein, a selective inhibitor of tyrosine kinases. In both primary astrocyte cultures and C6 glioma cells, the presence of genistein prevented both lipopolysaccharide- and cytokine-induced NOS activity in a dose-dependent manner. The presence of tyrphostin-25 (10 µ M ), which is highly specific for tyrosine kinases, also blocked iNOS induction. Additional characterization showed that genistein blocked iNOS induction in a dose-dependent manner (IC₅₀ of ∼ 40 µ M ), that the continuous presence of genistein was not necessary to observe inhibition, and that preincubation with genistein led to higher levels of inhibition than the simultaneous addition of genistein and inducers. The decrease in iNOS activity due to genistein was accompanied by a decrease in iNOS mRNA level as detected by a specific PCR assay. These results indicate that induction of astroglial iNOS expression requires tyrosine kinase activity.     

Peroxidase Activity of Hemoglobin��Haptoglobin Complexes: COVALENT AGGREGATION AND OXIDATIVE STRESS IN PLASMA AND MACROPHAGES*

As a hemoprotein, hemoglobin (Hb) can, in the presence of H₂O₂, act as a peroxidase. In red blood cells, this activity is regulated by the reducing environment. For stroma-free Hb this regulation is lost, and the potential for Hb to become a peroxidase is high and further increased by inflammatory cells generating superoxide. The latter can be converted into H₂O₂ and feed Hb peroxidase activity. Haptoglobins (Hp) bind with extracellular Hb and reportedly weaken Hb peroxidase activity. Here we demonstrate that: (i) Hb peroxidase activity is retained upon binding with Hp; (ii) in the presence of H₂O₂, Hb·Hp peroxidase complexes undergo covalent cross-linking; (iii) peroxidase activity of Hb·Hp complexes and aggregates consumes reductants such as ascorbate and nitric oxide; (iv) cross-linked Hb·Hp aggregates are taken up by macrophages at rates exceeding those for noncovalently cross-linked Hb·Hp complexes; (v) the engulfed Hb·Hp aggregates activate superoxide production and induce intracellular oxidative stress (deplete endogenous glutathione and stimulate lipid peroxidation); (vi) Hb·Hp aggregates cause cytotoxicity to macrophages; and (vii) Hb·Hp aggregates are present in septic plasma. Overall, our data suggest that under conditions of severe inflammation and oxidative stress, peroxidase activity of Hb·Hp covalent aggregates may cause macrophage dysfunction and microvascular vasoconstriction, which are commonly seen in severe sepsis and hemolytic diseases.As a hemoprotein, Hb,² in the presence of oxidizing equivalents such as H₂O₂, can act as a peroxidase with very high oxidizing potential (1). In red blood cells, this potentially dangerous activity is strictly regulated by the reducing environment and the lack of oxidizing equivalents. The inadvertently appearing ferric forms of Hb are short-lived, and the hemoprotein is effectively converted into ferro-Hb (deoxy-Hb) by metHb reductase (2). Normally, less than 2% of total Hb exists in the form of MetHb because the rate of Hb reduction is far greater than its oxidation (2). For stroma-free Hb, however, this intracellular regulation is lost, and the likelihood for Hb to act as a peroxidase is high. This possibility is markedly increased in the course of severe inflammation (e.g. in sepsis) by the generation of superoxide radicals by immune cells. The latter can be spontaneously or catalytically (by extracellular superoxide dismutase) converted into H₂O₂, a fuel for Hb peroxidase activity. In line with this, several clinical and experimental investigations have established that lethality in sepsis is increased in the setting of hemolysis (3–5).Circulating haptoglobin (Hp) provides an important endogenous defense against the toxic effects of Hb (6, 7). The major biological function of this abundant plasma protein is binding and recycling of stroma-free Hb via the macrophage CD163 receptor-mediated pathway (8, 9). It has been proposed that Hp possesses antioxidant activity and diminishes oxidative stress induced by stroma-free Hb (10–12). The antioxidant action of Hp toward Hb has been associated, at least in part, with weakening of its peroxidase activity (10) or preventing oxidation and cross-linking of Hb (7).Previous work has demonstrated that peroxidase activity of different hemoproteins, including Hb, can induce protein self-oxidation leading to covalent cross-linking and aggregation (13–16). Whether these hetero-oligomeric covalent aggregates retain the peroxidase activity is unknown. If the aggregates retain peroxidase activity, they may continue to be a source of oxidative stress both in circulation as well as in phagocytizing cells involved in their clearance such as macrophages.In the current work, we determined the extent to which: (i) Hb peroxidase activity is decreased by binding with Hp; (ii) peroxidase activity of Hb·Hp complexes initiates cross-linking into covalent hetero-oligomers; (iii) peroxidase activity of Hb·Hp complexes and aggregates utilizes nitric oxide (NO^•); (iv) Hb·Hp aggregates are taken up by macrophages as compared with noncovalent Hb·Hp complexes; and (v) the engulfed Hb·Hp aggregates induce oxidative stress and cytotoxicity. Here, we report that Hb·Hp complexes and aggregates are potent peroxidases capable of inducing oxidative stress in both plasma and macrophages. We further demonstrate the presence of Hb·Hp aggregates in septic plasma.     

Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na⁺, K⁺-ATPase activity, α₁ and β₁ subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na⁺, K⁺-ATPase α₁ subunit abundance increased in IR24 and IR48 cortical tissue and β₁ subunit decreased in IR48. In IR24 medullary tissue, α₁ abundance increased and returned to control values in IR48 while β₁ abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in α₁ mRNA in cortex and medulla that normalised after 24 h of reperfusion. β₁ mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult α₁ and β₁ protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na⁺, K⁺-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.     

Inflammatory bowel disease: Therapeutic limitations and prospective of the stem cell therapy

Inflammatory bowel disease(IBD), consisting primarily of ulcerative colitis and Crohn's disease, is a group of debilitating auto-immune disorders, which also increases the risk of colitis-associated cancer. However, due to the chronic nature of the disease and inconsistent treatment outcomes of current anti-IBD drugs(e.g., approximately 30% non-responders to anti-TNFα agents), and related serious side effects, about half of all IBD patients(in millions) turn to alternative treatment options. In this regard, mucosal healing is gaining acceptance as a measure of disease activity in IBD patients as recent studies have correlated the success of mucosal healing with improved prognosis. However, despite the increasing clinical realization of the significance of the concept of mucosal healing, its regulation and means of therapeutic targeting remain largely unclear. Here, stemcell therapy, which uses hematopoietic stem cells or mesenchymal stem cells, remains a promising option. Stem cells are the pluripotent cells with ability to differentiate into the epithelial and/or immune-modulatory cells. The overreaching concept is that the stem cells can migrate to the damaged areas of the intestine to provide curative help in the mucosal healing process. Moreover, by differentiating into the mature intestinal epithelial cells, the stem cells also help in restoring the barrier integrity of the intestinal lining and hence prevent the immunomodulatory induction, the root cause of the IBD. In this article, we elaborate upon the current status of the clinical management of IBD and potential role of the stem cell therapy in improving IBD therapy and patient's quality of life.