Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii

Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5??-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The K _m and k _cat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s^?1, respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

BPR1K653, a novel Aurora kinase inhibitor, exhibits potent anti-proliferative activity in MDR1 (P-gp170)-mediated multidrug-resistant cancer cells

Background

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.

Principal Findings

BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.

Conclusions and Significance

BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments.     

Design of synthetic hexapeptide substrates for prostate-specific antigen using single-position minilibraries.

Prostate-specific antigen (PSA), a serine endoprotease with chymotrypsin-like substrate specificity, is a marker used widely for detection of prostate cancer and other prostate diseases, catalyzing hydrolysis of the gel-forming proteins semenogelins I and II, which are synthesized and secreted by the seminal vesicle. In this study we report the use of two single-position minilibraries and RP-HPLC selection to optimize a hexapeptide substrate for PSA, spanning substrate positions P3 to P3'. PSA has been shown previously to prefer tyrosine in position P1 [Denmeade et al. (1997) Cancer Research, 57, 4924-4930]. Here we demonstrate preference for serine in position P1' and strong preference for phenylalanine in position P2. Based on these results we have designed and demonstrated the utility of the optimized fluorogenic PSA substrate 7-methoxy-coumarin-4-acetylGlnPheTyrSerSerAsnLys(epsilon-2,4-dinit rophenyl)amide, 1, which permits continuous monitoring of PSA endopeptidase activity at high sensitivity.     

Construction of expanded infant life tables: A method based on a new mortality law

A mortality law for infants is developed for constructing augmented-period infant life tables. For advanced countries, the three-parameter model is found to fit the mortality data for subdivisions of the first year of life extremely well, with estimates of the life-table functions calculated from the proposed model closely matching those constructed by the traditional method. Thus, considerable benefit is derived by replacing a large accumulation of data, marred by inevitable irregularities and errors arising from incomplete reporting and other sources, with a simple formula that can produce life-table functions at any age point and for any age interval and can be handled with some facility. We have also demonstrated how the model is used to calculate a number of new life-table functions and parameters of the lifetime distribution with their attendant applications, which include determination of the maximum life expectancy and the force of mortality at birth as well as certain average lifetimes for the stationary population. It is found that over 97% of the information contained in an augmented infant life table can be summarized by three parameters, with the remaining portion captured by another three parameters.     

Localization of the photoreceptor gene ROM1 to human chromosome 11 and mouse chromosome 19: sublocalization to human 11q13 between PGA and PYGM.

Rom-1 is a retinal integral membrane protein that, together with the product of the human retinal degeneration slow gene (RDS), defines a photoreceptor-specific protein family. The gene for rom-1 (HGM symbol: ROM1) has been assigned to human chromosome 11 and mouse chromosome 19 by Southern blot analysis of somatic cell hybrid DNAs. ROM1 was regionally sublocalized to human 11p13-11q13 by using three mouse-human somatic cell hybrids; in situ hybridization refined the sublocalization to human 11q13. Analysis of somatic cell hybrids suggested that the most likely localization of ROM1 is in the approximately 2-cM interval between human PGA (human pepsinogen A) and PYGM (muscle glycogen phosphorylase). ROM1 appears to be a new member of a conserved syntenic group whose members include such genes as CD5, CD20, and OSBP (oxysterol-binding protein), on human chromosome 11 and mouse chromosome 19. Localization of the ROM1 gene will permit the examination of its linkage to hereditary retinopathies in man and mouse.