Spectroscopic investigations, quantum chemical, drug likeness and molecular docking studies of methyl 1-methyl-4-nitro-pyrrole-2-carboxylate: A novel ovarian cancer drug.

Density functional theory (DFT) calculation was used to analyse the structural and vibrational properties of Methyl 1-Methyl-4-nitro-pyrrole-2-carboxylate (MMNPC) using the cc-pVTZ basis set. The potential energy surface scan and the most stable molecular structure were optimized using Gaussian 09 program. A potential energy distribution calculation was used to calculate and assign vibrational frequencies using the VEDA 4.0 program package. The Frontier Molecular Orbitals (FMOs) were analysed to determine their related molecular properties. Ab initio density functional theory (B3LYP/cc-pVTZ) method with basis set was used to calculate 13C NMR chemical shift values of MMNPC in the ground state. Fukui function and molecular electrostatic potential (MEP) analysis confirmed the bioactivity of the MMNPC molecule. The charge delocalization and stability of the title compound were studied using natural bond orbital analysis. All experimental spectral values from FT-IR, FT-Raman, UV-VIS, and 13C NMR are in good agreement with the value calculated by the DFT. Molecular docking analysis was carried out to find the MMNPC compound that can be used as a potential drug development candidate for ovarian cancer.

Spectroscopic characterization, DFT studies, molecular docking and cytotoxic evaluation of 4-nitro-indole-3-carboxaldehyde: A potent lung cancer agent.

The 4-nitro-1H-indole-carboxaldehyde (NICA) molecule was characterized experimentally using FT-IR, FT-Raman and UV-Vis spectra, and it was studied theoretically using DFT calculations. The optimized structure of the NICA molecule was determined by DFT calculations using B3LYP functional with cc-pVTZ basis set. The electron localization function (ELF) and local orbital localizer (LOL) studies were performed to visualize the electron delocalization in the molecule. The experimental and theoretical wavenumbers of the title molecule were assigned using VEDA 4.0 program. The charge delocalization and stability of the title molecule were investigated using natural bond orbital (NBO) analysis. Frontier molecular orbitals (FMOs) and related molecular properties were calculated. UV-Vis spectrum was calculated theoretically and validated experimentally. The reactive sites of the molecule were studied from the MEP surface and Fukui function analysis. The molecular docking analysis reveals that the NICA ligand shows better inhibitory activity against RAS, which causes lung cancer. The in vitro cytotoxic activity of the molecule against human lung cancer cell lines (A549) was determined by MTT assay. Thus, the NICA molecule can be used as a potential candidate for the development of the drug against lung cancer.

IFN-γ induction by neutrophil-derived IL-17A homodimer augments pulmonary antibacterial defense.

The role of interleukin-17A (IL-17A) in host defense against Legionella pneumophila remains elusive. To address this issue, we used Il17a(-/-), Il17f(-/-), and Il17a/Il17f(-/-) mice on a C57Bl/6 (non-permissive) background and IL-17 neutralizing Abs in mice on an A/J (permissive) background. Higher bacterial (L. pneumophila) counts in the lung and blood along with reduced neutrophil recruitment were detected in Il17a(-/-), but not Il17f(-/-), mice. We found that neutrophils produce IL-17A homodimer (IL-17A) during L. pneumophila infection, and hematopoietic cell-derived IL-17A is known to be important for bacterial clearance. Thus, intratracheal administration of wild-type neutrophils or recombinant IL-17A restored bacterial clearance and neutrophil recruitment in Il17a(-/-) mice. Furthermore, neutrophil-depleted Rag2(-/-) and Rag2/Il-2rγ(-/-) mice exhibited increased bacterial burden, reduced neutrophil influx and IL-17A production in the lung. Recombinant IFN-γ administration in Il17a(-/-) mice augmented bacterial elimination, whereas IL-17A administration in Ifnγ(-/-) mice did not augment bacterial clearance. IFN-γ is produced by T cells, but not neutrophils or macrophages, suggesting that neutrophil-derived IL-17A induces IFN-γ in a paracrine fashion. Human pneumonic lungs and human neutrophils challenged with L. pneumophila exhibited increased numbers of IL-17A producing cells. These findings display a novel function of neutrophil-derived IL-17A in antibacterial defense via the induction of IFN-γ in a paracrine manner.

NLRP12 modulates host defense through IL-17A-CXCL1 axis.

We used an extracellular pathogen Klebsiella pneumoniae to determine the role of NLRP12 (NOD-like receptor (NLR) family pyrin domain containing 12) as this bacterium is associated with devastating pulmonary infections. We found that human myeloid cells (neutrophils and macrophages) and non-myeloid cells (epithelial cells) show upregulation of NLRP12 in human pneumonic lungs. NLRP12-silenced human macrophages and murine Nlrp12(-/-) macrophages displayed reduced activation of nuclear factor-κB and mitogen-activated protein kinase, as well as expression of histone deacetylases following K. pneumoniae infection. NLRP12 is important for the production of interleukin-1ß (IL-1ß) in human and murine macrophages following K. pneumoniae infection. Furthermore, host survival, bacterial clearance, and neutrophil recruitment are dependent on NLRP12 following K. pneumoniae infection. Using bone marrow chimeras, we showed that hematopoietic cell-driven NLRP12 signaling predominantly contributes to host defense against K. pneumoniae. Intratracheal administration of either IL-17A+ CD4 T cells or chemokine (C-X-C motif) ligand 1 (CXCL1+) macrophages rescues host survival, bacterial clearance, and neutrophil recruitment in Nlrp12(-/-) mice following K. pneumoniae infection. These novel findings reveal the critical role of NLRP12-IL-17A-CXCL1 axis in host defense by modulating neutrophil recruitment against this extracellular pathogen.

N-[3-(4-Fluoro-benz-yl)-2,4-dioxo-1,3-diaza-spiro-[4.5]dec-8-yl]-2-methyl-benzene-sulfonamide.

In the title compound, C(22)H(24)FN(3)O(4)S, the cyclo-hexane ring adopts a chair conformation and the five-membered ring is essentially planar, with a maximum deviation of 0.040 (2) Å. The dihedral angles between the five-membered ring and the tolyl and fluoro-benzene rings are 56.74 (12) and 89.88 (12)°, respectively. The two terminal benzene rings make a dihedral angle of 63.53 (12)°. The crystal structure displays inter-molecular C-H⋯O and N-H⋯O hydrogen bonds. An intra-molecular C-H⋯O hydrogen bond also occurs.

N-{3-[2-(4-Fluoro-phen-oxy)eth-yl]-2,4-dioxo-1,3-diaza-spiro-[4.5]decan-7-yl}-4-meth-oxy-benzene-sulfonamide.

In the title compound, C(23)H(26)FN(3)O(6)S, the two terminal aromatic rings form a dihedral angle of 49.26 (12)°. The cyclo-hexane ring adopts a chair conformation and the five-membered ring is essentially planar, with a maximum deviation from planarity of 0.0456 (19) Å. The dihedral angles between the five-membered ring and the meth-oxy-benzene and fluoro-benzene rings are 33.56 (11) and 81.94 (12)°, respectively. The crystal structure displays N-H⋯O hydrogen bonds as well as weak inter-molecular C-H⋯O inter-actions.

Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.

Pasteurella (Mannheimia) haemolytica leukotoxin-induced cytolysis of bovine leukocytes: role of arachidonic acid and its regulation.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether AA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells were used as an experimental system to investigate the Lkt-induced [(3)H] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis toxin, inhibitors of cytosolic phospholipase A(2)(cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, Western blot analysis revealed the presence of G(i), G(s)and G(q)type G-proteins. These results demonstrate that AA metabolites from cPLA(2)activation contribute to Lkt-induced cytolysis and G(i)type G-proteins, Ca(2+)and PKC, regulate the cPLA(2)activity.

Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-kappaB activation and calcium elevation.

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity.

Tourniquet failure and arterial calcification. Case report and theoretical dangers.

A patient with Mönckeberg's calcinosis is presented in whom a pneumatic limb tourniquet failed to be effective because of calcification of the femoral artery wall. Bleeding from the operation site was noticed to be appreciably greater while the tourniquet was inflated since the cuff, though not occluding the femoral artery, acted as a very effective venous tourniquet. Theoretical risks which might be associated with the use of tourniquets in the presence of arterial calcification are fracture of the calcified vessel wall and systemic overdose of local anaesthetic agent following attempted regional intravenous block. The problem of tourniquet failure in general, and the dangers which might be associated with the use of tourniquets in patients with incompressible arteries, are briefly discussed.

Endometrial histology and progesterone levels in women using norethindrone acetate implants for contraception.

PIP: The effect of a single implant-D containing 40 mg of norethindrone acetate on the endometrium and serum progesterone, and its mode of contraceptive action was investigated. 20 women aged 20-40 (parity 1-5) had used implant-D for periods of 7-27 months. Endometrial biopsies and peripheral venous bloods were collected between the 16th-28th days of the menstrual cycle. Endometrial dating was done according to the criteria of Noyes et al. 19 of the 20 biopsies taken showed the endometrium in the secretory phase, and serum progesterone levels above 3 ng/ml confirming that ovulation had occurred. The biopsy of the remaining woman showed proliferative endometrium and serum progesterone of 2.15 ng/ml. The dating of the endometrium revealed disparity between stromal, glandular, and vascular components of the specimens; thus, only approximate dates could be assigned to a particular specimen. In the dating of the endometrium, 5 women showed retardation by more than 3 days and 4 women showed enhancement of 3 or more days. The results show that the norethindrone-acetate-containing silastic implant-D does not inhibit ovulation and 1 mode of its interaction in fertility control is by bringing about an imbalance in endometrial maturation during the luteal phase.^ieng