Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

Background  

Mass spectrometry has become a powerful tool for the analysis of large numbers of proteins in complex samples, enabling much of proteomics. Due to various analytical challenges, so far no proteome has been sequenced completely. O'Shea, Weissman and co-workers have recently determined the copy number of yeast proteins, making this proteome an excellent model system to study factors affecting coverage.     

Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee

Background  

Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues.     

The four hexamerin genes in the honey bee: structure,molecular evolution and function deduced from expression patterns in queens,workers and drones

Background  

Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens.     

Mutations on the Switch III region and the alpha3 helix of Galpha16 differentially affect receptor coupling and regulation of downstream effectors

Background

Gα₁₆ can activate phospholipase Cβ (PLCβ) directly like Gα_q. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα₁₆. Previous studies on Gα_q have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα₁₆ might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.

Results

Alanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα₁₆. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα₁₆ was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G₁₆ and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.

Conclusion

The integrity of the switch III region and α3 helix of Gα₁₆ is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα₁₆ to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G₁₆. The studied region was shown to bear multiple functionally important roles of G₁₆.     

New steroidal aromatase inhibitors: Suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death

Background  

Aromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.     

Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer

Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor. This highly controllable system combines short contact times between yeast and wort with the reduction of off-flavors to concentrations below threshold values. In the present study, the influence of immobilization of yeast to DEAE-cellulose on sugar fermentation and aldehyde reduction was monitored. Immobilized cells showed higher activities of hexokinase and pyruvate decarboxylase compared to cells grown in batch culture. In addition, a higher glucose flux was observed, with enhanced excretion of main fermentation products, indicating a reduction in the flux of sugar used for biomass production. ADH activity was higher in immobilized cells compared to that in suspended cells. However, during prolonged production a decrease was observed in NAD-specific ADH activity, whereas NADP-specific activity increased in the immobilized cells. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.     

Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

Background  

Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target.     

Application of Direct Agglutination Test (DAT) and Fast Agglutination Screening Test (FAST) for sero-diagnosis of visceral leishmaniasis in endemic area of Minas Gerais,Brazil

Background  

The direct agglutination test (DAT) has proved to be a very important sero-diagnostic tool combining high levels of intrinsic validity and ease of performance. Otherwise, fast agglutination screening test (FAST) utilises only one serum dilution making the test very suitable for the screening of large populations.