Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.     

Cytologic and histologic findings in multiple renal hybrid oncocytic tumors in a patient with Birt-Hogg-Dubé syndrome: a case report

BACKGROUND: Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant neoplastic syndrome characterized by multiple skin lesions, lung cysts and renal tumors. A variety of histologic types of renal tumors have been reported, including clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, oncocytoma and a recently described hybrid oncocytic tumor, which is thought to be highly associated with BHD. CASE: We report a case of a 48-year-old woman with BHD who initially presented to our institution with spontaneous pneumothorax and was found to have multiple lung cysts and renal tumors on computed tomography. We describe the fine needle aspiration findings of one of the renal tumors, which was suggestive of so-called hybrid oncocytic tumor. We also describe the gross and histologic findings of the multiple kidney tumors that the patient subsequently had excised. CONCLUSION: When multiple kidney tumors from a single patient appear oncycytic on fine needle aspiration, especially when focal clear cells are present, the possibility of oncocytomas and hybrid tumors associated with BHD must be entertained.     

Solvent free microwave synthesis and evaluation of antimicrobial activity of pyrimido[4,5-b]- and pyrazolo[3,4-b]quinolines

A series of 2-oxopyrimido[4,5-b]-, 2-thio[4,5-b]-, 1-(p-tosyl)pyrazolo[3,4-b]- and 1-(2',4'-dinitrophenyl)pyrazolo[3,4-b]-quinolines have been synthesized in good to excellent yields by environmentally benign solvent free microwave-induced techniques involving the condensation of 2-chloro-3-formylquinolines with urea, thiourea, p-toluenesulfonylhydrazide and 2,4-dinitrophenylhydrazine, respectively, using PTSA as a catalyst. All the synthesized compounds were evaluated for their antibacterial and antifungal activities. Most of the compounds showed the best activity against Escherichia coli and Pseudomonas aeruginosa.     

Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.     

Isolation, Characterization and Biological Role of Camelysin from Bacillus thuringiensis Subsp. israelensis

The present study reports a simple rapid method for isolating the zinc-containing metalloprotease camelysin from Bacillus thuringiensis subsp. israelensis (Bti) by extraction from intact bacterial cells with egg l-α-phosphatidylcholine containing monolamellar liposomes, followed by separation on a sucrose gradient. Characterization of the isolated camelysin revealed a molecular weight of 23 kDa and a pI of 6.2. The camelysin exhibited maximal activity against the substrate azocasein at a temperature of 37°C and pH 7.5. However, the enzyme’s activity remained high also at basic pH values (8–10). In a rich growth medium (LB), camelysin appeared at the late logarithmic phase of Bti growth and reached its maximum in the stationary phase. Camelysin was shown to activate the protoxins Cyt1Aa and Cyt2Ba produced by Bti. The hemolytic activity of Cyt1Aa increased from 40 to 70% and that of Cyt2Ba from 6 to 50% in the presence of 50% (w/w) camelysin. It is concluded that these protoxins can be activated not only by insect gut proteases, but also by the endogeneous metalloprotease camelysin of the Bti bacterium.     

Intensity Correlation-Based Calibration of FRET

Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes.