Some 3-(4-Aminophenyl)pyrrolidine-2,5-diones as All- trans -retinoic Acid Metabolising Enzyme Inhibitors (RAMBAs)

A series of (±) -3-(4-aminophenyl) pyrrolidin-2,5-diones substituted in the 1-, 3- or 1,3- position with an aryl or long chain alkyl function are weak inhibitors of the metabolism of all-trans retinoic acid (RA) by rat liver microsomes (68-75% inhibition) compared with ketoconazole (85%). Further studies with the 1-cyclohexyl analogue (1) (IC 50 = 98.8 μM, ketoconazole, 22.15 μM) showed that it was not stereoselective in its inhibition. (±) - (1) was not an inhibitor of pig brain microsomal enzyme (ketoconazole, IC 50 = 20.9 μM), had little effect on human liver microsomal enzyme (19.3%, ketoconazole, 81.6%) or human placental microsomal enzyme (9.8%, ketoconazole 73.9%) but was a weak inhibitor of human and rat skin homogenates (52.6% and IC 50 = 211.6 μM respectively; ketoconazole, 38.8% and 85.95 μM). In RA-induced cell cultures of human male genital fibroblasts and HaCat cells, (±) - (1) was a weak inhibitor (c. 53% at 200 μM) whereas ketoconazole showed high potency (c. 65% at 0.625 μM and 0.25 μM respectively). The nature of the induced target enzyme is discussed.     

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.     

Design, synthesis, and biological evaluation of callophycin A and analogues as potential chemopreventive and anticancer agents

Callophycin A was originally isolated from the red algae Callophycus oppositifolius and shown to mediate anticancer and cytotoxic effects. In our collaborative effort to identify potential chemopreventive and anticancer agents with enhanced potency and selectivity, we employed a tetrahydro-β-carboline-based template inspired by callophycin A for production of a chemical library. Utilizing a parallel synthetic approach, 50 various functionalized tetrahydro-β-carboline derivatives were prepared and assessed for activities related to cancer chemoprevention and cancer treatment: induction of quinone reductase 1 (QR1) and inhibition of aromatase, nitric oxide (NO) production, tumor necrosis factor (TNF)-α-induced NFκB activity, and MCF7 breast cancer cell proliferation. Biological results showed that the n-pentyl urea S-isomer 6a was the strongest inducer of QR1 with an induction ratio (IR) value of 4.9 at 50 μM [the concentration to double the activity (CD)=3.8 μM] and its corresponding R-isomer 6f had an IR value of 4.3 (CD=0.2 μM). The isobutyl carbamate derivative 3d with R stereochemistry demonstrated the most potent inhibitory activity of NFκB, with the half maximal inhibitory concentration (IC(50)) value of 4.8 μM, and also showed over 60% inhibition at 50 μM of NO production (IC(50)=2.8 μM). The R-isomer urea derivative 6j, having an appended adamantyl group, exhibited the most potent MCF7 cell proliferation inhibitory activity (IC(50)=14.7 μM). The S-isomer 12a of callophycin A showed the most potent activity in aromatase inhibition (IC(50)=10.5 μM).     

Synthesis and biological evaluation of novel N,N'-bis-methylenedioxybenzyl-alkylenediamines as bivalent anti-Alzheimer disease ligands

A novel series of N,N'-bis-methylenedioxybenzyl-alkylenediamines 5a-5g have been designed, synthesized and evaluated as bivalent anti-Alzheimer's disease ligands. The enzyme inhibition assay results indicated that compounds 5e-5g inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the micromolar range (IC(50), 2.76-4.24 μM for AChE and 3.02-5.14 μM for BuChE), which was in the same potential as the reference compound rivastigmine (IC(50), 5.50 μM for AChE and 1.60 μM for BuChE). It was found that compounds could bind simultaneously to the peripheral and catalytic sites of AChE. β-Amyloid (Aβ) aggregation inhibition assay results showed that compound 5e exhibited highest self-mediated Aβ fibril aggregation inhibition activity (40.3%) with a similar potential as curcumin (41.6%). It was also found that 5e-5g did not affect neuroblastoma cell viability at the concentration of 50 μM.     

Synergistic effects of amyloid peptides and lead on human neuroblastoma cells

Aggregated amyloid peptides (AP), major components of senile plaques, have been considered to play a very important and crucial role in the development and neuro-pathogenesis of Alzheimer's disease (AD). In the present in vitro, study the synergistic effects of Pb(2+), a heavy metal, and AP on the human neuroblastoma SH-SY5Y cells were investigated. The cells treated with Pb(2+) (0.01-10 μM) alone exhibited a significant decrease in viability and IC(50) was 5 μM. A similar decrease in viability was also observed when the cells were exposed to AP, Aβ1-40 (20-120 μM) and Aβ25-35 (2.5-15 μM) for 48 hrs. The IC(50) values were 60 μM and 7.5 μM for Aβ1-40 and Aβ25-35 respectively. To assess the synergistic effects the cells were exposed to IC(50) of both AP and Pb(2+), which resulted in further reduction of the viability. The study was extended to determine the lactate dehydrogenase (LDH) release to assess the cytotoxic effects, 8-isoprostane for extent of oxidative damage, COX 1 and 2 for inflammation related changes, p53 protein for DNA damage and protein kinases A and C for signal transduction. The data suggest that the toxic effects of AP were most potent in the presence of Pb(2+), resulting in an aggravated clinical pathological condition. This could be attributed to the oxidative stress, inflammation neuronal apoptosis and an alteration in the activities of the signaling enzymes.     

Development and evaluation of multifunctional agents for potential treatment of Alzheimer's disease: application to a pyrimidine-2,4-diamine template

We investigated a group of 2-benzylpiperidin-N-benzylpyrimidin-4-amines with various electron-withdrawing or electron-donating groups (EWGs or EDGs, respectively) as multi-targeted Alzheimer's disease (AD) therapeutics. The synthesized derivatives were screened for anti-cholinesterase (AChE and BuChE), anti-Aβ-aggregation (AChE- and self-induced) and anti-β-secretase (BACE-1) activities in an effort to identify lead, multifunctional candidates as part of our multi-targeted approach to treat AD. Biological assessment revealed that the nature of the substituent on the C-4 benzylamine group (e.g., halogen vs methoxy-based) greatly affected the biological profile. In vitro screening identified N(2)-(1-benzylpiperidin-4-yl)-N(4)-(3,4-dimethoxybenzyl)pyrimidine-2,4-diamine (7h) as the lead candidate with a dual ChE (AChE IC(50)=9.9 μM; BuChE IC(50)=11.4 μM), Aβ-aggregation (AChE-induced=59.3%; self-induced=17.4% at 100 μM) and BACE-1 (34% inhibition at 10 μM) inhibitory profile along with good cell viability (% neuroblastoma cell viability at 40 μM=81.0%). Molecular modeling studies indicate that a central pyrimidine-2,4-diamine ring serves as a suitable template to develop novel small molecule candidates to target multiple pathological routes in AD.     

Antifungal Activities of Different Extracts of Marine Macroalgae Against Dermatophytes and Candida Species

Algae are bioactive natural resources, and due to the medical importance of superficial mycoses, we focused the action of macroalgae extracts against dermatophytes and Candida species. Seaweed obtained from the Riacho Doce beach, Alagoas (Brazil), were screened for the antifungal activity, through crude extracts using dichloromethane, chloroform, methanol, ethanol, water and chloroform and hexane fractions of green, brown and red algae in assays with standard strains of the dermatophytes Trichophyton rubrum, T. tonsurans, T. mentagrophytes, Microsporum canis, M. gypseum and yeasts Candida albicans, C. krusei, C. guilliermondi and C. parapsilosis. The M44-A and M27-A2/M38A manuals by CLSI were followed, and the minimum inhibitory concentration (MIC) ranged from 0.03 to 16.00 μg ml(-1), and an inhibition halo of 10.00-25.00 mm was observed for dermatophytes, while for yeast, it was from 8.00 to 16.00 μg ml(-1) and 10.00-15.00 mm. M. canis showed MIC of 0.03 μg ml(-1) and the largest inhibition halo in T. rubrum (25.00 mm) through the use of the methanol extract. For C. albicans, dichloromethane, methanol and ethanol extracts formed the largest inhibition halo. The ethanol extract was shown to be the best inhibiting fungi growth, and chloroform and hexane fractions of H. musciformis inhibited the growth of all dermatophytes and C. albicans, yielding the conclusion that apolar extracts obtained from algae presented the best activity against important pathogenic fungi.     

An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC(50), 7.2±1.8 μM), human breast adenocarcinoma (MCF-7) (IC(50), 10.0±0.5 μM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC(50), 6.0±1 μM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC(50), 5.8±0.3 μM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC(50), 6.5±1μM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 μM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3±1.8 μM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i)) of 5.2±1.5 μM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.     

Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Adaptation of dihydrofolate reductase (DHFR)‐deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time‐consuming and labor‐intensive process because nonadapted DHFR‐deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension‐adapted DHFR‐deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2–359 μM enhanced cell growth in in‐house CDM. Addition of 3.6 μM THF to in‐house CDM resulted in a more than 2.5‐fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9‐fold enhancement of maximum viable cell density. An anchorage‐ and serum‐dependent DHFR‐deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in‐house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension‐adapted DHFR‐deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR‐deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539–1546, 2016     

Nonenzymatic amperometric response of glucose on a nanoporous gold film electrode fabricated by a rapid and simple electrochemical method

An enzyme-free amperometric method was established for glucose detection using a nanoporous gold film (NPGF) electrode prepared by a rapid one-step anodic potential step method within 5 min. The prepared NPGF had an extremely high roughness and was characterized by scanning electron microscopy (SEM) and cyclic voltammetry. Electrochemical responses of the as-prepared NPGF to glucose in 0.1M phosphate buffer solution (PBS, pH 7.4) with or without Cl(-) were discussed. In amperometric studies carried out at -0.15 V in the absence of Cl(-), the NPGF electrode exhibited a high sensitivity of 232 μA mM(-1)cm(-2) and gave a linear range from 1mM up to 14 mM with a detection limit of 53.2 μM (with a signal-to-noise ratio of 3). In addition, the oxidation of ascorbic acid (AA) and uric acid (UA) can be completely eliminated at such a low applied potential. On the other hand, the quantification of glucose in 0.1M PBS (pH 7.4) containing 0.1M NaCl offered an extended linear range from 10 μM to 11 mM with a sensitivity of 66.0 μA mM(-1)cm(-2) and a low detection limit of 8.7 μM (signal-to-noise ratio of 3) at a detection potential of 0.2V.     

Development of 2-substituted-N-(naphth-1-ylmethyl) and N-benzhydrylpyrimidin-4-amines as dual cholinesterase and Aβ-aggregation inhibitors: Synthesis and biological evaluation

A group of 2-substituted N-(naphth-1-ylmethyl)pyrimidin-4-amines (6a-k) and N-benzhydrylpyrimidin-4-amines (7a-k) in conjunction with varying steric and electronic properties at the C-2 position were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. The naphth-1-ylmethyl compound 6f (2-(4-cyclohexylpiperazin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine) exhibited optimum dual ChE (AChE IC(50)=8.0 μM, BuChE IC(50)=3.9 μM) and hAChE-promoted Aβ-aggregation inhibition (30.8% at 100 μM), whereas in the N-benzhydryl series, compound 7f (N-benzhydryl-2-(4-cyclohexylpiperazin-1-yl)pyrimidin-4-amine) exhibited optimum combination of dual ChE (AChE IC(50)=10.0 μM, BuChE IC(50)=7.6μM) and hAChE-promoted Aβ-aggregation inhibition (32% at 100 μM). These results demonstrate that a 2,4-disubstituted pyrimidine ring serves as a suitable template to target multiple pathological routes in AD, with a C-2 cyclohexylpiperazine substituent providing dual ChE inhibition and potency whereas a C-4 diphenylmethane substituent provides Aβ-aggregation inhibition.     

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth (Micropterus salmoides) bass

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 μm and width of 246.1 μm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) μm and width of 6.9 ± 1.1 (4.0-9.0) μm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 μm and width of 148.1 μm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) μm and width of 10.6 ± 0.6 (9.0-11.8) μm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P = 0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P = 0.001) or fall (P = 0.008). Prevalence and seasonal differences were not determined for M. micropterii .     

Taurine uptake by cultured human lymphoblastoid cells

Cultured human lymphoblastoid cells take up taurine from the medium by two processes: 1) a temperature-dependent, Na⁺-dependent, saturable “active”-transport system and 2) diffusion. The active transport has properties similar to those reported for taurine transport by other tissues. Apparent K_m is about 25 μM and V_max about 7.2 pmol/min/10⁶ cells; saturation occurs at 100 μM taurine. Uptake is competitively inhibited by the β-amino acids hypotaurine (50% inhibition at 44 μM) and β-alanine (50% at 152 μM), as measured at 50 μM taurine. Taurocyamine inhibits 50% at 260 μM. Chlorpromazine and imipramine are strong uncompetitive inhibitors, giving 50% inhibition at 26 μM and 115 μM, respectively; at these concentrations cellular viability per se is not affected. Ouabain inhibits 40–50% over a concentration range of 4–500 μM. Diffusion of taurine into the cells is proportional to concentration up to 20 mM. However, at the concentration of taurine in human plasma, 40–100 μM, active transport would provide 90% of the taurine taken up.     

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (-)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 μM and 110 μM, respectively). β-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 μM). Neither menthol nor β-nicotyrine was an inactivator of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 μM, kinact was 0.61 min(-1)). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.     

Inhibition of Parasite Protein Kinase C by New Antileishmanial Imidazolidin-2-one Compounds

The protein kinase C (PKC) family of isoenzymes mediate a wide range of signal transduction pathways in many different cells lines. Little is known regarding the presence and functional roles of PKC in Leishmania spp. Here we report the inhibition of parasite PKC by new imidazolidinone compounds. The most active derivative 7 showed an important activity (IC 50 =9.9 μM) against the clinical relevant stage of parasites in comparison with Glucantime ® (IC 50 =464.5 μM), without inducing toxicity on human fibroblast cells (IC 50 =102 μM). Pretreatment of intact parasites with 10 μM of compound 7 inhibited 80% of PKC activity. At the same concentration, this compound inhibited 70% of the parasite-host cell invasion process. An in vivo model showed that compound 7 reduced the liver parasite burden by 25% and spleen parasite burden by 44%. These results provide the first evidence that PKC plays a critical role in the invasion process. Thus Leishmania PKC activity could be a relevant therapeutic target and the imidazolidinones novel antileishmanial candidates.     

In Vitro Micropropagation and Flowering in Artemisia annua

A protocol for quick regeneration of a large number of plantlets of Artemisia annua (source of a potent antimalarial drug) is being reported. Multiple shoots were obtained in large numbers from juvenile as well as vegetative parts of mature plant on Murashige and Skoog’s medium (MS) having 3% sucrose and 800 μM myoinositol and supplemented with NAA (0.5 μM) + BAP (13.0 μM) + GA₃ (0.3 μM) + Asp (300.0 μM) + Glu (700.0 μM) + Arg (300.0 μM) + Cys - HCl (30.0 μM). Reversal of reproductive to vegetative phase and back to reproductive phase could be achieved in the cultured flower buds. The shoots obtained on the above medium could be induced to flower. In addition, new shoots that differentiated from vegetative parts of juvenile and mature explants also produced flowers when cultured on MS with GA₃ (0.3 μM). Since artemisinin estimation is correlative to flowering, our results would facilitate better understanding of biosynthesis of this drug in vitro.     

Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 μM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 μM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.     

Effect of the K channel openers NIP-121 and cromakalim on melittin-induced relaxation of guinea-pig isolated trachea

We have investigated the effect o f the K⁺ channel openers (+)-7,8- dihydro-6,6-dimethyl-7-hydroxy-8-(2-oxo-piperidin-1-yl)-6H-pyrano[2,3f]benz-2, 1,3-oxadiazole (NIP-121) and cromakalim on the relaxation induced by the phospholipase A2 activator melittin in guinea-pig isolated trachea. Melittin (0.1 to 3.0 μg/ml caused concentration-dependent relaxation of tracheal spirals precontracted with LTD₄. The magnitude of relaxation was about 40% of that obtained by 1 mM aminophylline. Melittin-induced relaxation was also observed in tracheas precontracted with histamine or the thromboxane A₂ mimetic U46619. The relaxation to melittin was prevented by the cyclooxygenase inhibitor indomethacin (5 μM) or by removal of the tracheal epithelium, suggesting that cyclooxygenase products, possibly dependent on the epithelium, may be implicated in the response to melittin. Pretreatment of tracheas with NIP-121 (0.03 μM) or cromakalim (0.4 μM) did not affect the contraction to LTD₄ but enhanced the relaxation to melittin. This enhancement to melittin was completely inhibited by the ATP-dependent K⁺ channel blocker glibenclamide (1 μM). Higher concentrations of NIP-121 (0.1 μM) or cromakalim (1 μM) did not enhance the response to melittin. In the presence of indomethacin, NIP-121 (0.03 μM) or cromakalim (0.4 μM) enhanced PGE₂ induced relaxation of tracheas precontracted with LTD₄. These results suggest that cyclooxygenase products, possibly dependent on the epithelium, may be involved in melittin-induced relaxation. The enhancement of relaxation to melittin by IK⁺ channel activation might be due, at least in part, to an increased.     

In vitro cormlet production of saffron (Crocus sativus L. Kashmirianus) and their flowering response under greenhouse

A complete protocol for the saffron cormlet production under in vitro conditions and subsequent flowering under greenhouse conditions is described. Highest number of cormlets (70.0 ± 0.30) per corm slice (explant) could be regenerated on Murashige and Skoog (MS) half strength medium supplemented with thidiazuron (TDZ) (20 μM), Indole acetic acid (IAA) (10 μM), and sucrose (40 g/l). Maximum germination (90%) of these cormlets could be achieved on MS medium containing 6-benzyl amino purine (BAP) (20 μM) and α-naphthalene acetic acid (NAA) (15 μM). In order to increase the size of the in vitro raised cormlets, these were cultured on MS medium containing TDZ (15 μM) and IAA in the range of 1.5-30 μM. Maximum increase in cormlet size could be attained on TDZ (15 μM) + IAA (12.5 μM) + sucrose (30 g/l), and the average size of cormlets was 2.5g. In another experiment, apical vegetative buds of actively growing corms were cultured for cormlet development, and corms of size 2.5g could be developed on MS medium with NAA (15 μM), BAP (20 μM), and sucrose (30 g/l). The in vitro developed cormlets were dried under shade at 25 ± 2°C for 7 d. These were then planted in small cups containing clay loam soil and kept in green house at 20 ± 2°C. In vitro developed cormlets with mean weight 2.5 g showed maximum flowering (25%) as well as vegetative growth (55%), while only 19% cormlets of 2.0 g flowered. To our knowledge this is the first report on successful flowering from in vitro raised cormlets under greenhouse.