Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.     

Nitric Oxide Synthase in Bovine Superior Cervical Ganglion

Abstract: We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by N ^G-methyl-L-arginine (NMA), and the inhibition was reversed by L-arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2',5'-ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca²⁺/calmodulin dependent and required NADPH and tetrahydrobiopterin as co-factors. Superoxide dismutase protected and oxyhemo-globin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155-kDa protein band. Immunohisto chemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine-β-hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine-induced stimulation of soluble guanylyl cyclase.     

Chain elongation of fatty acid in brain: a comparison of mitochondrial and microsomal enzyme activities

The activities of mitochondrial and microsomal fatty acid-elongating enzymes have been measured in rat brain during postnatal development and in brains of jimpy, msd, and quaking mice. The microsomal enzyme activity rose from a low in the immature brain to a maximum at 21 days of age and then declined to low levels in the mature brain. The developmental patterns were similar for all acyl-CoAs tested. The maximum activity fell sharply from C₁₆ to C₁₈ and then fell gradually with increase in fatty acid chain length up to C₂₄. The activities for monounsaturated acyl-CoAs were slightly higher than for corresponding saturated esters. The mitochondrial enzyme activity was high in the immature brain and remained virtually unchanged during further brain development. This activity steadily decreased with increasing chain length from C₁₆ to C₂₄. The microsomal enzyme activity was reduced in myelin-deficient mutants compared to their controls. The extent of reduction was most severe for C₂₀- to C₂₄-CoAs followed by C₁₈-CoA and then C₁₆-CoA, for which the activity was reduced only in the jimpy mouse. The activities for C₂₀- to C₂₄-CoAs in jimpy, msd, and quaking mice were 12, 38, and 52% of the control, respectively. The mitochondrial enzyme activity was not affected by these mutations. Fatty acid synthetase activity was similar in the mutant and control mice. These results suggest that the deficiency of long-chain fatty acids in the central nervous system of myelin-deficient mouse mutants is due to reduced synthesis by the microsomal enzyme, which is directly related to myelination. The brain mitochondrial enzyme appears to be unrelated to myelination.     

Regulation of prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts by ascorbic acid

Studies with confluent human skin fibroblasts maintained in 0.5% serum supplemented medium have given new insight into the regulatory influences of ascorbate. These include a reduction of prolyl hydroxylase activity, a stimulation of lysyl hydroxylase activity, and an acceleration of collagen production. The lack of parallel between prolyl hydroxylase activity and collagen production indicates that the rate of collagen synthesis is not controlled by the level of prolyl hydroxylase.     

Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP gamma S greater than ATP greater than AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATP gamma S-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATP gamma S after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATP gamma S retains enzyme activation after membrane washing. These results suggest either that ATP gamma S stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATP gamma S pretreatment.     

N omega-nitro-L-arginine: a potent inhibitor of the L-arginine-dependent soluble guanylate cyclase activation pathway in LLC-PK1 cells

Oxytocin increased cyclic GMP levels in LLC-PK1 porcine kidney epithelial cells through activation of soluble guanylate cyclase. NG-Monomethyl-L-arginine and N omega-nitro-L-arginine inhibited oxytocin (10 microM) induced cyclic GMP accumulation with IC50 values of 2.3 microM and 140 nM, respectively, and the inhibition was prevented with L-arginine. Both inhibitors at 100 microM lowered the basal levels of cyclic GMP, but did not affect those induced by 1 microM sodium nitroprusside and 100 nM atrial natriuretic factor. These data support our hypothesis that an endothelium-derived relaxing factor-like substance is formed as the endogenous activator of soluble guanylate cyclase in an L-arginine-dependent fashion in various cell types. N omega-Nitro-L-arginine is 16 times more potent than NG-monomethyl-L-arginine as a specific inhibitor of this pathway in LLC-PK1 cells.     

Trichomonas vaginalis: identification of a triacylglycerol acylhydrolase

This work describes the identification of a triacylglycerol lipase named TVLip directly onto blood-LB-agar plates by hemolytic screening of a Trichomonas vaginalis cDNA expression library. Sharing significant similarity in the primary sequence with other lipases, the theoretical 3D structure of the TVLip was resolved. The structure reveals the predictive conserved characteristics of other lipases from EC3.1.1.3 group, although presenting one amino acid change in the catalytic triad Ser-His-Asp. Finally, analysis of Northern blot indicates that the expression of the TVLip gene is up-regulated by iron.     

Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production

Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.