Differentially Expressed RNA from Public Microarray Data Identifies Serum Protein Biomarkers for Cross-Organ Transplant Rejection and Other Conditions

Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers.     

Sequence specific 1H, 13C and 15N resonance assignments of hahellin in 8 M urea

The sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of hahellin in 8 M urea-denatured state have been accomplished by NMR spectroscopy. Secondary chemical shift analysis reveals the native-like propensities for β-rich conformation in the denatured state.     

Fractal binding and dissociation kinetics of heart-related compounds on biosensor surfaces

A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg white apoA-I rHDL immobilized on a biosensor chip surface (1), native, mildly oxidized, and strongly oxidized LDL in solution to a heparin-modified Au-surface of a surface plasmon resonance (SPR) biosensor (2), and TRITC-labeled HDL in solution to a bare optical fiber surface (3). Single-and dual-fractal models were used to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (4). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.     

Detection of glucose and related analytes by biosensors: a fractal analysis

A fractal analysis is used to model the binding and dissociation kinetics of connective tissue interstitial glucose, adipose tissue interstitial glucose, insulin, and other related analytes on biosensor surfaces. The analysis provides insights into diffusion-limited analyte-receptor reactions occurring on heterogeneous biosensor surfaces. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of heterogeneity or roughness [fractal dimension (D(f))] present on the biosensor chip surface. The binding and dissociation rate coefficients are sensitive to the degree of heterogeneity on the surface. For example, for the binding of plasma insulin, as the fractal dimension value increases by a factor of 2.47 from D(f1)=0.6827 to D(f2)=1.6852, the binding rate coefficient increases by a factor of 4.92 from k(1)=1.0232 to k(2)=5.0388. An increase in the degree of heterogeneity on the probe surface leads to an increase in the binding rate coefficient. A dual-fractal analysis is required to fit the binding kinetics in most of the cases presented. A single fractal analysis is adequate to describe the dissociation kinetics. Affinity (ratio of the binding to the dissociation rate coefficient) values are also presented. Interferents for glucose, such as uric acid and ascorbic acid, were also detected by using glucose biosensors based on carbon nanotube (CNT) nanoelectrode ensembles (NEEs) (Lin Y, Lu F, Tu Y, Ren Z).     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Molecular mechanisms associated with the regulation of apoptosis by the two alternatively spliced products of c-Myb

The c-myb proto-oncogene encodes two alternatively spliced mRNAs, which in turn code for proteins of 75 kDa and 89 kDa. It is at present unclear whether the two isoforms of c-Myb perform identical functions or whether they mediate different biological effects. To assess their role in apoptotic death of hematopoietic cells, we expressed the two isoforms of c-Myb in the murine myeloid cell lines 32Dcl3 and FDCP1. Our results show that while ectopic overexpression of p75 c-Myb results in the acceleration of cell death, similar overexpression of p89 c-Myb results in the protection of cells from apoptotic death. An analysis of gene expression changes with mouse cDNA expression arrays revealed that while p75 c-Myb blocked the expression of glutathione S-transferase micro mRNA, p89 c-Myb greatly enhanced the expression of this gene. These results were further confirmed by Northern blot analysis. Ectopic overexpression of the glutathione S-transferase micro gene in 32Dcl3 cells resulted in protection of cells from interleukin-3 withdrawal-induced cell death similar to that seen with the ectopic overexpression of p89 c-Myb. These results suggest that the two isoforms of c-Myb differentially regulate apoptotic death of myeloid cells through differential regulation of glutathione S-transferase micro gene expression.     

Computational identification of putative programmed translational frameshift sites

MOTIVATION: In an effort to identify potential programmed frameshift sites by statistical analysis, we explore the hypothesis that selective pressure would have rendered such sites underabundant and underrepresented in protein-coding sequences. We developed a computer program to compare the frequencies of k-length subsequences of nucleotides with the frequencies predicted by a zero order Markov chain determined by the codon bias of the same set of sequences. The program was used to calculate and evaluate the distribution of 7-base oligonucleotides in the 6000+ putative protein-coding sequences of S. cerevisiae preliminary to the laboratory testing of the most highly underrepresented oligos for frameshifting efficiency. RESULTS: Among the most significant results is the finding that the heptanucleotides CUU-AGG-C and CUU-AGU-U, sites of the programmed +1 translational frameshifts required for the production in yeast of actin filament-binding protein ABP140 and telomerase subunit EST3, respectively, rank among the least represented of phase I heptanucleotides in the coding sequences of S. cerevisiae. Laboratory experiments demonstrated that other underrepresented heptanucleotides identified by the program, for example GGU-CAG-A, are also prone to significant translational frameshifting, suggesting the possibility that genes containing other underrepresented heptamers may also encode transframe products. AVAILABILITY: The program is available for download from http://www.gesteland.genetics.utah.edu/freqAnalysis SUPPLEMENTARY INFORMATION: Complete results from the analysis of S. cerevisiae are available on http://www.gesteland.genetics.utah.edu/freqAnalysis     

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.