Use of micelles in studying drug-binding sites: simulation of the tubulin-bound fluorescence of colchicine

Enhancement of the fluorescence intensity of colchicine occurs in media of low polarity and appreciable viscosity; this is suggested to be the basis of the intensification of its fluorescence when it is bound to and immobilized in tubulin. We show here that the tubulin-bound fluorescence features of colchicine are largely reconstructed upon solubilizing it in chosen micellar aggregates that offer optimal polarities and microviscosities. Triton X-100 and bile salt micelles intensify the colchicine emission but the maximal effects are obtained with tetrameric aggregates of the peptide melittin. Estimates of the polarity, microviscosity and binding-site dimensions of colchicine are obtained using this mimetic approach. Our results suggest that well chosen micellar systems act as good models to reconstruct and analyze the spectral properties of molecules immobilized in their binding sites.     

An in vitro demonstration of peroxisome proliferation and increase in peroxisomal β-oxidation system mRNAs in cultured rat hepatocytes treated with ciprofibrate

Using the normal adult rat hepatocytes, plated on rat tail collagen-coated dishes and fed a chemically defined medium, we demonstrate here that ciprofibrate at 0.1 mM concentration, increases significantly the mRNA levels of fatty acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and thiolase (the three enzymes of the β-oxidation system), and causes peroxisome proliferation. Increase in mRNA levels of these genes was evident within 1 h and was maximal 24 h after the addition of ciprofibrate. In hepatocytes cultured in the absence of ciprofibrate, the basal levels of these enzymes were low and further declined with time. Concomitant treatment of hepatocytes with cycloheximide did not inhibit or superinduce the mRNA levels, indicating that this induction may represent a primary (direct) effect of this compound on the expression of these genes and does not apparently involve short-lived repressor protein(s).     

Lipids of Cuscuta reflexa and changes in lipids of its host plants after infection

The lipid level (fresh weight basis) of Cuscuta reflexa Roxb. was related to the lipid content of the host plants Meilicago saliva L., Helianthus annuus L., Pisum sativum L. and Lantana camara L. Parasitizing by the dodder significantly increased the total lipid level of the hosts. The increase was mainly due to enhancement in the neutral lipid fraction.
The level of phospholipid in the parasite was always higher than in its hosts. Phospholidyl choline and phosphatidyl ethanolamine constituted about 65% of the total phospholipid of Cuscuta. This was followed by phosphatidyl inositol (ca 20%) and phosphatidyl glycerol (ca 12%). Phosphatidic acid constituted only ca 3% of the phospholipids of Cuscuta. Although the total phospholipid levels of various host plants were not affected as a result of the infection by Cuscuta, a significant decrease occurred in the levels of phosphatidyl eholine and phosphatidyl ethanolamine as well as marked increases in phosphatidyl inositol and phosphatidic acid. The infected tissue showed an increase in phospholipase D activity as compared with the controls. The results have been discussed in relation to changes in permeability of the infected tissue.     

Cissogenin,a pregnane genin from Marsdenia tenacissima

A new polyhydroxy pregnane designated as cissogenin has been isolated from the seeds of Marsdenia tenacissima. Its structure has been established as 3β,11α,12β,14β,20 S-pentahydroxy-pregn-5-ene.     

Probiotic Attributes and Inhibitory Effects of Lactobacillus plantarum MYS84 against the Growth and Biofilm Formation of Pseudomonas aeruginosa

Microbiology - Manufacture of fermented foods/products, which, apart from basic nutrition, have health-promoting effects, is flourishing. Within the field of fermented foods, rapidly expanding is...     

Proteomic profiling of serum samples from chikungunya-infected patients provides insights into host response

Background

Chikungunya is a highly debilitating febrile illness caused by Chikungunya virus, a single-stranded RNA virus, which is transmitted by Aedes aegypti or Aedes albopictus mosquito species. The pathogenesis and host responses in individuals infected with the chikungunya virus are not well understood at the molecular level. We carried out proteomic profiling of serum samples from chikungunya patients in order to identify molecules associated with the host response to infection by this virus.

Results

Proteomic profiling of serum obtained from the infected individuals resulted in identification of 569 proteins. Of these, 63 proteins were found to be differentially expressed (≥ 2-fold) in patient as compared to control sera. These differentially expressed proteins were involved in various processes such as lipid metabolism, immune response, transport, signal transduction and apoptosis.

Conclusions

This is the first report providing a global proteomic profile of serum samples from individuals infected with the chikungunya virus. Our data provide an insight into the proteins that are involved as host response factors during an infection. These proteins include clusterin, apolipoproteins and S100A family of proteins.     

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks

Background

SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream^® instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results.

Results

We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters.

Conclusion

The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.     

Testing effects of signal transduction pathways on cadherin junctional complex assembly using quantitative image analysis

Cadherin adhesion molecules function in numerous cell biological processes that influence embryo development, normal cell physiology, and pathophysiology of many disease processes. Cadherins nucleate the assembly of the adherens junction, a cell-to-cell adhesion plaque that is prominent in simple epithelial cells and found in many cell types. Numerous cell biological approaches have been used to study this interesting class of molecules. Here, we outline methodology used in our studies of junctional complexes to examine effects of signaling molecules on assembly mechanisms. This is a quantitative method that allows the investigator to test the combined effect of two different signaling processes to determine whether these two signals act in concert within the same pathway. We discuss how this method could be generalized to other studies to examine consequences of various experimental manipulations on the assembly of cellular structures.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.