RB status as a determinant of response to UCN-01 in non-small cell lung carcinoma. (65/3151)

7-Hydroxystaurosporine (UCN-01), a protein kinase inhibitor in clinical development, demonstrates potent antineoplastic activity. To determine whether specific genetic abnormalities would modulate the response to UCN-01, a model of human non-small cell lung carcinoma (NSCLC) cell lines with differential abnormalities of p16CDKN2, RB, and p53 was used for these studies. Cell growth was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and cell cycling was studied using flow cytometric analysis of DNA content. Changes in protein levels and phosphorylation were assessed by Western blotting. In cell lines expressing wild-type RB (A549 and Calul), UCN-01 treatment resulted in dose-dependent growth inhibition, arrest of cells in G1, and a reduction of cells in S phase. p16CDKN2-null cells showed similar growth inhibition to normal fetal lung fibroblasts. UCN-01-induced growth arrest was accompanied by induction of p21CDKN1 and a shift of Rb to the hypophosphorylated state in both p53 wild-type and mutant cell lines. In contrast, UCN-01 treatment of the RB-null cell line H596 resulted in less growth inhibition. To test the role of RB in response to UCN-01, effects of treatment were examined in two human isogenic models of RB expression: the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). In the Rb-expressing 5637 subline (RB5), UCN-01 treatment resulted in Rb hypophosphorylation and an accumulation in G1 in contrast to the parent line. Similarly, the wild-type Rb-expressing DU-145 sublines (DU1.1 and B5) showed increased G1 arrest compared with the parent cells. We conclude that UCN-01-induced G1 arrest can occur in cells null for p53 and p16CDKN2, and that RB status influences the ability of UCN-01 to induce a G1 arrest. These data suggest that the molecular profile of cell cycle regulating genes in individual tumors may predict responsiveness and provide insight into optimal therapeutic application of this new antineoplastic agent.  (+info)

Plant products as antimicrobial agents. (66/3151)

The use of and search for drugs and dietary supplements derived from plants have accelerated in recent years. Ethnopharmacologists, botanists, microbiologists, and natural-products chemists are combing the Earth for phytochemicals and "leads" which could be developed for treatment of infectious diseases. While 25 to 50% of current pharmaceuticals are derived from plants, none are used as antimicrobials. Traditional healers have long used plants to prevent or cure infectious conditions; Western medicine is trying to duplicate their successes. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. This review attempts to summarize the current status of botanical screening efforts, as well as in vivo studies of their effectiveness and toxicity. The structure and antimicrobial properties of phytochemicals are also addressed. Since many of these compounds are currently available as unregulated botanical preparations and their use by the public is increasing rapidly, clinicians need to consider the consequences of patients self-medicating with these preparations.  (+info)

Diversity and distribution of nicotinic acetylcholine receptors in the locus ceruleus neurons. (67/3151)

The neurons of the locus ceruleus are responsible for most of the noradrenergic innervation in the brain and nicotine potentiates noradrenaline release from their terminals. Here we investigated the diversity and subcellular distribution of nicotinic acetylcholine receptors (nAChRs) in the locus ceruleus both somatically, by combining single-cell reverse transcription-PCR with electrophysiological characterization, and at the level of nerve terminals, by conducting noradrenaline efflux experiments. The proportion of neurons in the locus ceruleus expressing the nicotinic subunit mRNAs varied from 100% (beta2) to 3% (alpha2). Yet, two populations of neurons could be distinguished on the basis of the pattern of expression of nAChR mRNAs and electrophysiological properties. One population (type A) of small cells systematically expressed alpha3 and beta4 mRNAs (and often alpha6, beta3, alpha5, alpha4), and nicotinic agonists elicited large currents with a potency order of cytisine > nicotine. Another population (type B) of cells with large soma did not contain alpha3 and beta4 mRNAs but, systematically, alpha6 and beta3 (and often alpha4) and responded to nicotinic agonists in the order of nicotine > cytisine. The nicotinic modulation of noradrenaline release in the hippocampus displayed an order of potency nicotine > cytisine, suggesting that noradrenergic terminals in the hippocampus originate largely from type B cells of the locus ceruleus. Accordingly, immunocytochemical labeling showed that beta3 is present in hippocampal terminals. The alpha6beta3beta2(alpha4) heterooligomer thus behaves as the main nicotinic regulator of the ceruleo-hippocampal pathway.  (+info)

Effects of Delphinium alkaloids on neuromuscular transmission. (68/3151)

The Delphinium alkaloids methyllycaconitine (MLA), nudicauline, 14-deacetylnudicauline (14-DN), barbinine, and deltaline were investigated for their effects on neuromuscular transmission in lizards. The substituent at C14 provides the only structural difference among the alkaloids MLA, nudicauline, 14-DN, and barbinine. Deltaline lacks the N-(methylsuccinyl)anthranilic acid at C18 common to the other four alkaloids. Each alkaloid reversibly reduced extracellularly recorded compound muscle action potential (CMAP) amplitudes in a concentration-dependent manner. The IC(50) values for CMAP blockade were between 0.32 and 13.2 microM for the N-(methylsuccinimido)anthranoyllycacotonine-type alkaloids and varied with the C14 moiety; the IC(50) value for deltaline was 156 microM. The slopes of the concentration-response curves for CMAP blockade were similar for each alkaloid except barbinine, whose shallower curve suggested alternative or additional mechanisms of action. Each alkaloid reversibly reduced intracellularly recorded spontaneous, miniature end-plate potential (MEPP) amplitudes. Alkaloid concentrations producing similar reductions in MEPP amplitude were 0.05 microM for 14-DN, 0.10 microM for MLA, 0.50 microM for barbinine, and 20 microM for deltaline. Only barbinine altered the time constant for MEPP decay, further suggesting additional or alternative effects for this alkaloid. MLA and 14-DN blocked muscle contractions induced by exogenously added acetylcholine. All five alkaloids are likely nicotinic receptor antagonists that reduce synaptic efficacy and block neuromuscular transmission. The substituent at C14 determines the potency and possibly the mechanism of nicotinic acetylcholine receptor blockade for MLA, nudicauline, 14-DN, and barbinine at neuromuscular synapses. The lower potency of deltaline indicates that the N-(methylsuccinyl)anthranilic acid at C18 affects alkaloid interactions with nicotinic acetylcholine receptors at neuromuscular junctions.  (+info)

Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II. (69/3151)

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.  (+info)

Inhibition of Candida rugosa lipase by berberine and structurally related alkaloids, evaluated by high-performance liquid chromatography. (70/3151)

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.  (+info)

Polycitone A, a novel and potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases. (71/3151)

Polycitone A, an aromatic alkaloid isolated from the ascidian Polycitor sp. exhibits potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. The drug inhibits retroviral reverse transcriptase (RT) [i.e. of human immunodeficiency virus type 1 (HIV), murine leukaemia virus (MLV) and mouse mammary tumour virus (MMTV)] as efficiently as cellular DNA polymerases (i.e. of both DNA polymerases alpha and beta and Escherichia coli DNA polymerase I). The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function, on the other hand, is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the substrate binding site, since this inhibitor behaves competitively with respect to dTTP with poly(rA).oligo(dT) as template primer. Chemical transformations of the five phenol groups of polycitone A by methoxy groups have a determinant effect on the inhibitory potency. Thus, the pentamethoxy derivative which is devoid of all hydroxy moieties, loses significantly, by 40-fold, the ability to inhibit the DNA polymerase function. Furthermore, this analogue lacks the ability to inhibit DNA primer extension as well as the formation of the RT-DNA complex. Indeed, inhibition of the first step in DNA polymerization, the formation of the RT-DNA complex, and hence, of the overall process, could serve as a model for a universal inhibitor of the superfamily of DNA polymerases.  (+info)

Oxidized low-density lipoprotein enhances myogenic tone in the rabbit posterior cerebral artery through the release of endothelin-1. (72/3151)

BACKGROUND AND PURPOSE: Cerebral arteries develop stretch-induced myogenic tone, which plays an important role in the regulation of blood flow to the brain. Although the effect of oxidized LDL (Ox-LDL) on many aspects of the vascular endothelial and smooth muscle cell function have been extensively investigated, its influence on myogenic activity has not been studied. METHODS: The effect of Ox-LDL on the myogenic tone that develops in the perfused rabbit posterior cerebral artery at intramural pressures between 40 and 90 mm Hg was examined. RESULTS: Ox-LDL (10 microg/mL) significantly enhanced myogenic tone by 21.4+/-6.1% to 28.5+/-1.8% at 60 to 90 mm Hg pressure (P<0.05) but had no influence on norepinephrine- (0.5 to 1 micromol/L) and KCl (20 mmol/L)-induced constriction. Ox-LDL was effective whether the artery was exposed to it from the intraluminal or the extraluminal surface. Lysophosphatidylcholine (10 micromol/L), a lipid component of Ox-LDL, had an equivalent potentiating effect. Native LDL (100 microg/mL) was inactive. The myogenic tone-potentiating effect of Ox-LDL was abolished by endothelium removal but was not influenced by the NO synthase inhibitor N(G)-nitro-L-nitro-arginine methyl ester (50 micromol/L). This effect was reversed by the endothelin-1 (ET-1) antagonist BQ-123 (1 micromol/L). This concentration blocked 1 to 3 nmol/L ET-1-induced constriction without altering constriction induced by 40 mmol/L KCl. The potentiating effect was suppressed by the specific protein kinase C inhibitor chelerythrine (1 micromol/L). CONCLUSIONS: Ox-LDL enhances myogenic tone through the release of ET-1 from the endothelium of the rabbit posterior cerebral artery.  (+info)