Synthesis, bioactivity, and cloning of the L-type calcium channel blocker omega-conotoxin TxVII.

omega-Conotoxin TxVII is the first conotoxin reported to block L-type currents. In contrast to other omega-conotoxins, its sequence is characterized by net negative charge and high hydrophobicity, although it retains the omega-conotoxin cysteine framework. In order to obtain structural information and to supply material for further characterization of its biological function, we synthesized TxVII and determined its disulfide bond pairings. Because a linear precursor with free SH groups showed a strong tendency to aggregate and to polymerize, we examined many different conditions for air oxidation and concluded that a mixture of cationic buffer and hydrophobic solvent was the most effective for the folding of TxVII. Synthetic TxVII was shown to suppress the slowly inactivating voltage-dependent calcium current in cultured Lymnaea RPeD1 neurons and furthermore to suppress synaptic transmission between these neurons and their follower cells. In contrast, TxVII did not block calcium flux through L-type channels in PC12 cells, suggesting a phyletic or subtype specificity in this channel family. Disulfide bond pairings of TxVII and its isomers were determined by enzymatic fragmentation in combination with chemical synthesis, thus revealing that TxVII has the same disulfide bond pattern as other omega-conotoxins. Furthermore, the CD spectrum of TxVII is similar to those of omega-conotoxins MVIIA and MVIIC. The precursor sequence of TxVII was determined by cDNA cloning and shown to be closest to that of delta-conotoxin TxVIA, a sodium channel inactivation inhibitor. Thus TxVII conserves the structural fold of other omega-conotoxins, and the TxVIA/TxVII branch of this family reveals the versatility of its structural scaffold, allowing evolution of structurally related peptides to target different channels.     

In vivo antipyretic,antiemetic, in vitro membrane stabilization,antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

Background

The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.

Results

In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC₅₀ value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC₅₀ value 216.77 μg/ml) cytotoxic activity.

Conclusions

Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.     

A microarray study of MPP<Superscript>+</Superscript>-treated PC12 Cells: Mechanisms of toxicity (MOT) analysis using bioinformatics tools

Background

This paper describes a microarray study including data quality control, data analysis and the analysis of the mechanism of toxicity (MOT) induced by 1-methyl-4-phenylpyridinium (MPP⁺) in a rat adrenal pheochromocytoma cell line (PC12 cells) using bioinformatics tools. MPP⁺ depletes dopamine content and elicits cell death in PC12 cells. However, the mechanism of MPP⁺-induced neurotoxicity is still unclear.

Results

In this study, Agilent rat oligo 22K microarrays were used to examine alterations in gene expression of PC12 cells after 500 μM MPP⁺ treatment. Relative gene expression of control and treated cells represented by spot intensities on the array chips was analyzed using bioinformatics tools. Raw data from each array were input into the NCTR ArrayTrack database, and normalized using a Lowess normalization method. Data quality was monitored in ArrayTrack. The means of the averaged log ratio of the paired samples were used to identify the fold changes of gene expression in PC12 cells after MPP⁺ treatment. Our data showed that 106 genes and ESTs (Expressed Sequence Tags) were changed 2-fold and above with MPP⁺ treatment; among these, 75 genes had gene symbols and 59 genes had known functions according to the Agilent gene Refguide and ArrayTrack-linked gene library. The mechanism of MPP⁺-induced toxicity in PC12 cells was analyzed based on their genes functions, biological process, pathways and previous published literatures.

Conclusion

Multiple pathways were suggested to be involved in the mechanism of MPP⁺-induced toxicity, including oxidative stress, DNA and protein damage, cell cycling arrest, and apoptosis.

     

Genetics of quantitative traits in Arabidopsis thaliana

The genetic control of 22 quantitative traits, including developmental rates and sizes, was examined in generations of Arabidopsis thaliana derived from the cross between the ecotypes, Columbia (Col) and Landsberg erecta (Ler). The data were obtained from three sets of families raised in the same trial: the 16 basic generations, that is, parents, F(1)'s, F(2)'s, backcrosses, recombinant inbred lines (RILs) and a triple test cross (TTC), the latter produced by crossing the RILs to Col, Ler and their F(1). The data were analysed by two approaches. The first (approach A) involved traditional generation mean and variance component analysis and the second (B), based around the RILs and TTC families, involved marker-based QTL analysis.From (A), genetic differences between Col and Ler were detected for all traits with moderate heritabilities. Height at flowering was the only trait to show heterosis. Dominance was partial to complete for all height traits, and there was no overdominance but there was strong evidence for directional dominance. For most other traits, dominance was ambidirectional and incomplete, with average dominance ratios of around 80%. Epistasis, particularly of the duplicate type that opposes dominance, was a common feature of all traits. The presence of epistasis must imply multiple QTL for all traits.The QTL analysis located 38 significant effects in four regions of chromosomes I, II, IV and V, but not III. QTL affecting rosette size and leaf number were identified in all four regions, with days to maturity on chromosomes IV and V. The only QTL for height was located at the expected position of the erecta gene (chromosome II; 50 cM), but the additive and dominance effects of this single QTL did not adequately explain the generation means. The possible involvement of other interacting height QTL is discussed.     

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.     

Aging of mesenchymal stem cell in vitro

Background  

A hot new topic in medical treatment is the use of mesenchymal stem cells (MSC) in therapy. The low frequency of this subpopulation of stem cells in bone marrow (BM) necessitates their in vitro expansion prior to clinical use. We evaluated the effect of long term culture on the senescence of these cells.     

Correction for Irrelevant Absorption in Multicomponent Spectrophotometric Assay of Riboflavin, Formylmethylflavin, and Degradation Products: Kinetic Applications

In the spectrophotometric assay of multicomponent systems involved in drug degradation studies, some minor or unknown degradation products may be present. These products may interfere in the assay and thus invalidate the results due to their absorption in the range of analytical wavelengths. This interference may be eliminated by the application of an appropriate correction procedure to obtain reliable data for kinetic treatment. The present study is based on the application of linear and non-linear irrelevant absorption corrections in the multicomponent spectrophotometric assay of riboflavin and formylmethylflavin during the photolysis and hydrolysis studies. The correction procedures take into account the interference caused by minor or unknown products and have shown considerable improvement in the assay data in terms of the molar balance. The treatment of the corrected data has led to more accurate kinetic results in degradation studies.     

Evaluation of (ɳ6-p-cymene) ruthenium diclofenac complex as anticancer chemotherapeutic agent: interaction with biomolecules,cytotoxicity assays

abstract

The designing of metal-based anticancer therapeutic agents can be optimized in a better and rapid way if the ligands utilized have standalone properties. Therefore, even when the organometallic/coordination complex (i.e., metallodrug) gets dissociated in extreme conditions, the ligand can endorse its biological properties. Herein, we have synthesized and characterized ?⁶-p-cymene ruthenium diclofenac complex. Furthermore, the ruthenium complex interactions with human serum albumin (HSA) and ct-DNA have been studied using various spectroscopic studies viz., UV, fluorescence, and circular dichroism and exhibited a significant binding propensity. Furthermore, in vitro cytotoxicity assays were carried out against human breast cancer “MCF-7” cell line. The ?⁶-p-cymene ruthenium diclofenac complex registered significant cytotoxicity with an IC₅₀ value of ～25.0?µM which is comparable to the standard drugs. The ?⁶-p-cymene ruthenium diclofenac complex was able to decrease the MCF-7 cell proliferation and induced significant levels of apoptosis with relatively low toxicity.