Reverse-phase High-pressure Liquid Research Articles

Rearranged oleananes: Structural identification and distribution in a worldwide set of Late Cretaceous/Tertiary oils

The three most abundant rearranged oleanoid triterpanes eluting early in gas chromatography (GC) have been isolated from a Niger Delta oil and characterized using nuclear magnetic resonance (NMR) spectroscopy. The NMR data confirmed the identification of two of them, observed previously in the literature, as 1(10 → 5)abeo-3β-methyl-24β-nor-18α(H)-oleanane and 5(4 → 3)abeo-3α(H), 5β(Η), 18α(Η)-oleanane on the basis of X-ray crystallography and comparison with a synthetic standard. The third compound was a novel rearranged oleanane, 3α,5β-dimethyl-23α,25-dinor-10β(H),18α(H)-oleanane, with one of the methyl groups at C-4 in the oleanane structure shifted to C-3 and another shifted from C-10 to C-5. The distribution of these three rearranged oleananes and other, yet unidentified, rearranged oleanoid C 30 triterpanes in 25 Late Cretaceous- or Tertiary-sourced oils from Angola, Canada, Greece, India, Indonesia, Iran, New Zealand, Nigeria and Vietnam was examined by way of reverse phase high pressure liquid chromatography (HPLC), GC–mass spectrometry (GC–MS) and GC–MS–MS. Mass spectra of rearranged oleananes and compounds tentatively assigned as rearranged taraxastanes are presented. Rearranged oleananes and other rearranged triterpanes are probably formed by dehydration and rearrangement of higher plant triterpenoids functionalized at C-3. The distribution of rearranged oleananes and their relative concentration seems to be dependent on the extent of clay catalysis during diagenesis. A diagenetic pathway for formation of the rearranged oleananes is proposed. All 25 oils have almost identical rearranged oleanane/oleanane and diasterane/regular sterane ratio values. Two unidentified early eluting (GC) rearranged oleananes are only abundant in oils with a high proportion of diasteranes. Bicadinanes were found in 20 of the 25 oils.

Use of reversed phase high pressure liquid cromatography for the physicochemical and thermodynamic characterization of oxyresveratrol/β-cyclodextrin complexes

Knowledge of the complexation process of oxyresveratrol with β-cyclodextrin (β-CD) under different physicochemical conditions is essential if this potent antioxidant compound is to be used successfully in both food and pharmaceutical industries as ingredient of functional foods or nutraceuticals, despite its poor stability and bioavailability. In this paper, the complexation of oxyresveratrol with natural CDs was investigated for first time using RP-HPLC and mobile phases to which α-, β-, and γ-CD were added. Among natural CDs, the interaction of oxyresveratrol with β-CD was more efficient than with α- and γ-CD. The decrease in the retention times with increasing concentrations of β-CD (0–4 mM) showed that the formation constants ( K F ) of the oxyresveratrol/β-CD complexes were strongly dependent on both the water–methanol proportion and the temperature of the mobile phase employed. However, oxyresveratrol formed complexes with β-CD with a 1:1 stoichiometry in all the physicochemical conditions tested. Moreover, to obtain information about the mechanism of the oxyresveratrol affinity for β-CD, the thermodynamic parameters Δ G° , Δ H° and Δ S° were obtained. Finally, to gain information on the effect of the structure of different compounds belonging to the stilbenoids family on the K F values, the complexation of other molecules, resveratrol, pterostilbene and pinosylvin, was studied and compared with the results obtained for the oxyresveratrol/β-CD complexes.

Orally administered doxycycline accumulates in synovial fluid compared to plasma

Tetracycline compounds have been used to slow the progression of osteoarthritis (OA) and rheumatoid arthritis but the concentration of doxycycline attained in synovial fluid following oral, low-dose administration has yet to be determined. To determine the concentration of doxycycline in synovial fluid following oral, low-dose administration. Six mature horses received doxycycline (5 mg/kg bwt q. 12 h for 5 doses). Venous blood and synovial fluid samples were collected at t=0, 0.25, 0.5, 1, 12, 24, 48 and 72 h. Doxycycline concentrations were measured using reverse phase high pressure liquid chromatography with ultraviolet detection. Doxycycline concentrations at all time points after t=0 were above the lower limit of quantification for the assay. Plasma concentrations of doxycycline were above 0.21 microg/ml at t=0.5 h. The mean+/-s.d. peak concentration (Cmax) of doxycycline in plasma was 0.37+/-0.22 microg/ml and time to peak concentration was 0.54+/-0.19 h. Synovial fluid concentrations of doxycycline were above 0.12 microg/ml 1 h after drug administration. The mean Cmax of doxycycline in the synovial fluid was 0.27+/-0.10 microg/ml. The penetration factor of doxycycline from plasma into synovial fluid, as determined by a ratio of the area-under-the-curve for synovial fluid:plasma during the sampling period, was 4.6. Orally administered doxycycline distributes easily into synovial fluid with a penetration factor of 4.6. Terminal half-life of the drug in synovial fluid was longer than in the plasma, indicating possible accumulation in this compartment. Further in vivo studies are warranted to define a medication protocol prior to routine clinical use of doxycycline for the treatment of OA.

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

Flow Cytometry, HPLC-RP, and metAFLP Analyses to Assess Genetic Variability in Somatic Embryo-Derived Plantlets of Gentiana pannonica Scop

Cytometric and molecular techniques were used to verify genetic uniformity among somatic embryo-derived plantlets of Gentiana pannonica Scop. Cytometric analysis of regenerants revealed absence of chromosomal changes and alterations in ploidy. However, reverse phase high pressure liquid chromatography detected higher levels of methylation in regenerated plants than those of control plants. These changes were further investigated using a quantitative molecular marker-based approach. This revealed that numerous tissue culture-induced variations, ∼3% (epi)mutations, were observed, including sequence variation and changes in methylation patterns. Moreover, complex patterns of variation, including combinations of genetic and epigenetic changes, were relatively high (ca. 9%). Overall, tissue culture-induced variation reached 16%; while, demethylation was lower than de novo methylation in heterozygotic material and similar in all regenerated plantlets.

Development and Validation of RP-HPLC Method for The Simultaneous Estimation of Amoxicillin Trihydrate and Bromhexine Hydrochloride from Oily Suspension

A new simple, rapid and precise reverse phase high pressure liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of amoxicillin trihydrate and bromhexine hydrochloride from oily suspension. An ODS C 18 (250 X 4.5mm ID), 5 μ particle size with mobile phase methanol and glacial acetic acid (50:50 v/v) were used. The flow rate was 1.0ml/min and responses were measured at 254 nm. The retention time for amoxicillin trihydrate and bromhexine hydrochloride were observed at 3.04 and 8.18 min. respectively. Linearity for amoxicillin trihydrate and bromhexine hydrochloride were in the range of 8-50 mcg/ml and 5-25 mcg/mL respectively. Percent recovery was 99.54% and 98.65% for amoxicillin trihydrate and bromhexine hydrochloride respectively. The proposed method can be applied for the routine analysis of amoxicillin trihydrate and bromhexine hydrochloride in combination.

A Rapid Determination of Cinnarizine in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid and precise reverse phase high pressure liquid chromatographic method has been developed for the estimation of cinnarizine from pharmaceutical formulation. The method was developed using MICRA-NPS C18(length×OD×ID =33×8.0×6.0 mm, 1.5 μm) column with a mobile phase consisting of acetonitrile, triethylamine buffer (adjusted to pH 4.5 with 10% w/v potassium hydroxide) and tetrahydrofuran in the ratio 30:66:4 respectively, at a flow rate of 0.5 mL/min. Wavelength was fixed at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the routine estimation of cinnarizine in pharmaceutical dosage form.

TMP21 Transmembrane Domain Regulates γ-Secretase Cleavage

TMP21 has been shown to be associated with the γ-secretase complex and can specifically regulate γ-cleavage without affecting ϵ-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of γ-secretase activity was investigated independent of ϵ-cleavage using an amyloid-β precursor proteinϵ (APPϵ) construct which lacks the amyloid intracellular domain domain. The APPϵ construct behaves similarly to the full-length precursor protein with respect to α- and β-cleavages and is able to undergo normal γ-processing. Co-expression of APPϵ and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Aβ-positive fragments, consistent with an inhibition of γ-secretase cleavage. The APPϵ system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the γ-secretase complex and could decrease γ-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Aβ production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased γ-cleavage activity.

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

Lipidomic Analysis of Dynamic Eicosanoid Responses during the Induction and Resolution of Lyme Arthritis

Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2(-/-) mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies.

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

Analysis and purification of O-decanoyl sucrose regio-isomers by reversed phase high pressure liquid chromatography with evaporative light scattering detection

Purification of seven regio-isomers of O-decanoyl sucrose, 2- O-, 3- O-, 4- O-, 6- O-, 3′- O-, 4′- O- and 6′- O-decanoyl sucrose, were performed by LC followed by preparative RP-HPLC with ELSD. Using an optimized gradient of acetonitrile in water 2- O-, 3- O-, 6- O- and 3′- O-decanoyl sucrose were purified in yields (w/w) of 52.5%, 34.7%, 45.0% and 36.9%, respectively. In the purified preparations of the 2- O- and 3′- O-decanoyl sucrose respectively, acyl migration was observed as a result of the drying process. Lyophilization resulted in the highest purities (w/w) of 96% and 100% for the 2- O- and the 3′- O-decanoyl sucrose, respectively.

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

alpha-Accessory factor (AAF) stimulates the activity of DNA polymerase-alpha.primase, the only enzyme known to initiate DNA replication in eukaryotic cells ( Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265, 13221-13230 ). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44.AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-alpha.primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-(3)H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-alpha.primase template affinity and stimulate both DNA primase and polymerase-alpha activities in vitro.

Phenolic compounds and antioxidant activities of some Hypericum species: A comparative study with H. perforatum

This study was designed to determine the polyphenolic contents of the extracts and to evaluate the antioxidant activity of Hypericum origanifolium Willd. and H. montbretii Spach. (Guttiferae (Hypericaceae)), The possible composition activity relationship was investigated and the results were compared with that of H. perforatum L. Methanol, ethyl acetate, and water were used as solvents to produce extracts from flowers and leaves of the plants. The determination of phenolic acids in the Hypericum species was achieved by using a modified Reverse phase-High pressure liquid chromatography (RP-HPLC) method adopting an internal standard. It was observed that chlorogenic and caffeic acids were higher in all extracts. The highest values were found in ethyl acetate extracts for total phenolic content as gallic acid and for the flavonoids and flavonols as rutin equivalents (all measurements are mg/g), respectively. Hypericum extracts were evaluated for their radical scavenging activity by the 2,2-diphenyl-1-picryhydrazyl radical (DPPH) and their oxidative stability by the Rancimat method. Results were compared with Butyllated hidroxy toluene (BHT), a synthetic antioxidant, and with a reference plant, H. perforatum. A good correlation between antioxidant activity and total phenol content in the extracts was observed. In an antioxidant activity assay, the leaf extracts of H. origanifolium were found to be two or three times more active than those of BHT, H. perforatum, and H. montbretii leaves and flowers. In an antiradical activity assay, leaves and flowers of H. montbretii and leaves of H. origanifolium were the most active at the tested concentrations, exhibiting an activity comparable to that of the positive control BHT, but all of the extracts, with the exception of the leaves of H. montbretii, showed activity weaker than the leaves and flowers of H. perforatum, the reference plant.

A Novel Anti‐inflammatory Compound, Artonkin‐4′‐O‐glucoside, from the Leaves of Artocarpus tonkinensis Suppresses Experimentally Induced Arthritis*

Artocarpus tonkinenesis (Moraceae) has been used in Vietnamese traditional medicine for the treatment of backache and joint diseases since many 100 years. We have previously shown that a crude extract of A. tonkinensis elicited anti-inflammatory effects in rat collagen-induced arthritis (CIA), with significant improvement of disease symptoms. However, the pharmacological basis of the bioactivity of A. tonkinensis extract is not known. In the present study, we have isolated four individual active components from A. tonkinensis extract by reverse phase high-pressure liquid chromatography. The structures of the compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and their biological effects investigated. A novel biologically active flavonoid glucoside (5-hydroxy-8-hydroxymethyl-8-methyl-2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenyl]-8H-pyrano[3,2-g]chromen-4-one) with an average molecular mass of 514.49 Da was isolated.We have named the compound artonkin-4'-O-glucoside. The name 'artonkin' for the novel flavonoid part of the compound was coined from the Latin name of its source Artocarpus tonkinensis. The three other active flavonoid glucosides isolated and characterized were alphitonin-4-O-beta-D-glucoside, maesopsin-4-O-beta-D-glucoside and kaempherol-3-O-beta-D-glucoside. All four compounds were found to cause anti-inflammatory effect with different potencies. The anti-inflammatory effects demonstrated in the rat model of arthritis correlate well with the inhibition of mitogen-induced T-cell proliferation. Furthermore, the compounds inhibit production of cytokines, such as tumour necrosis factor-a and interferon-c, in mitogen-stimulated T cells in a concentration-dependent manner. We postulate that the isolated flavonoids suppress T-cell proliferation as well as cytokine expression and thereby contribute to an amelioration of arthritis severity in CIA.

Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration

Two non-toxic PLA2s were purified to homogeneity from Cerastes cerastes Tunisian snake venom. The purification process employed gel filtration on Sephadex G-75 followed by C18 reverse phase high-pressure liquid chromatography. These two acidic enzymes, namely CC-PLA2-1 and CC-PLA2-2, have a molecular weight of 13,737.52 and 13,705.63 Da, respectively. These two PLA2 are the first reported glycosylated phospholipases A2 purified from snake venom. The rates of glycosylation are 2.5% and 0.5% (w/w), respectively. Specific activities of 1800 U/mg and 2400 U/mg for CC-PLA2-1 and CC-PLA2-2, respectively, were measured at optimal conditions. CC-PLA2-1 and CC-PLA2-2 strongly inhibited coagulation. They also exhibited a marked dose-dependent inhibitory effect on platelet aggregation induced by ADP and arachidonic acid in platelet-rich plasma. Interestingly, CC-PLA2-1 and CC-PLA2-2 inhibited in a dose-dependent manner adhesion of IGR39 melanoma and HT1080 fibrosarcoma cells to fibrinogen and fibronectin. Furthermore, both CC-PLA2-1 and CC-PLA2-2 abolished HT1080 cell migration towards fibrinogen and fibronectin. This activity is reported for the first time for PLA2 enzymes.

Polo-like Kinase 2 (PLK2) Phosphorylates α-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.

Cytotoxic activity of nemorosone in neuroblastoma cells

Neuroblastoma is the second most common solid tumour during childhood, characterized by rapid disease progression. Most children with metastasized neuroblastoma die despite intensive chemotherapy due to an intrinsic or acquired chemotherapy resistance. Thus, new therapeutic strategies are urgently needed. Here, we demonstrate that the novel compound nemorosone isolated from alcoholic extracts of Clusia rosea resins by reverse phase high pressure liquid chromatography (RP-HPLC) exerts cytotoxic activity in neuroblas-toma cell lines both parental and their clones selected for resistance against adriamycin, cisplatin, etoposide or 5-fluorouracil. Cell cycle studies revealed that nemorosone induces an accumulation in G0/G1- with a reduction in S-phase population combined with a robust up-regulation of p21Cip1. Furthermore, a dose-dependent apoptotic DNA laddering accompanied by an activation of caspase-3 activity was detected. Nemorosone induced a significant dephosphorylation of ERK1/2 in LAN-1 parental cells probably by the inhibition of its upstream kinase MEK1/2. No significant modulation of signal transducers JNK, p38 MAPK and Akt/PKB was detected. The enzymatic activity of immunoprecipitated Akt/PKB was strongly inhibited in vitro, suggesting that nemorosone exerts its anti-proliferative activity at least in part by targeting Akt/PKB in the cell lines studied. In addition, a synergistic effect with Raf-1 inhibitor BAY 43-9006 was found. Finally, nemorosone induced a considerable down-regulation of N-myc protein levels in parental LAN-1 and an etoposide resistant sub-line at the same drug-concentrations.

The Biflavonoid Isoginkgetin Is a General Inhibitor of Pre-mRNA Splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

All mature tRNA molecules have the conserved CCA sequence at the 3' end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 3' end. Using Escherichia coli tRNA(Cys) as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA(Cys), separated from the unlabeled precursor tRNA by reverse phase high-pressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNA(Cys) with E. coli CysRS reveals an equilibrium K(d) closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyC-labeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.