Pilot and feasibility study: comparative proteomic analysis by 2-DE MALDI TOF/TOF MS reveals 14-3-3 proteins as putative biomarkers of response to neoadjuvant chemotherapy in ER-positive breast cancer

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity? Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Flavobacterium johnsoniae SprA is a cell surface protein involved in gliding motility

Flavobacterium johnsoniae cells glide rapidly over surfaces by an unknown mechanism. Transposon-induced sprA mutants formed nonspreading colonies on agar, and the cells examined in wet mounts were deficient in attachment to surfaces and were almost completely nonmotile. Exposure of intact cells to proteinase K cleaved the 270-kDa SprA into several large peptides, suggesting that it is partially exposed on the cell surface.     

Visualization and quantification of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> intraerythrocytic merozoites

Malaria, a leading parasitic killer, is caused by Plasmodium spp. The pathology of the disease starts when Plasmodium merozoites infect erythrocytes to form rings, that matures through a large trophozoite form and develop into schizonts containing multiple merozoites. The number of intra-erythrocytic merozoites is a key-determining factor for multiplication rate of the parasite. Counting of intraerythrocytic merozoites by classical 2-D microscopy method is error prone due to insufficient representation of merozoite in one optical plane of a schizont. Here, we report an alternative 3-D microscopy based automated method for counting of intraerythrocytic merozoites in entire volume of schizont. This method offers a considerable amount of advantages in terms of both, ease and accuracy.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.     

Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar×Swarna RILs

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. 168 F(7) RILs derived from Madhukar×Swarna were used to map QTLs for iron and zinc concentrations in unpolished rice grains. Iron ranged from 0.2 to 224ppm and zinc ranged from 0.4 to 104ppm. Genome wide mapping using 101 SSRs and 9 gene specific markers showed 5 QTLs on chromosomes 1, 3, 5, 7 and 12 significantly linked to iron, zinc or both. In all, 14 QTLs were identified for these two traits. QTLs for iron were co-located with QTLs for zinc on chromosomes 7 and 12. In all, ten candidate genes known for iron and zinc homeostasis underlie 12 of the 14 QTLs. Another 6 candidate genes were close to QTLs on chromosomes 3, 5 and 7. Thus the high priority candidate genes for high Fe and Zn in seeds are OsYSL1 and OsMTP1 for iron, OsARD2, OsIRT1, OsNAS1, OsNAS2 for zinc and OsNAS3, OsNRAMP1, Heavy metal ion transport and APRT for both iron and zinc together based on our genetic mapping studies as these genes strictly underlie QTLs. Several elite lines with high Fe, high Zn and both were identified.     

Exploiting whole genome sequence data to fine map and characterize candidate genes within a quantitative trait loci region affecting androstenone on porcine chromosome 5

Male piglets are routinely castrated to eliminate boar taint. However, this treatment is undesirable, and alternative approaches, including genetic strategies to reduce boar taint, are demanded. Androstenone is one of the causative agents of boar taint, and a QTL region affecting this pheromone has previously been reported on SSC5: 22.6–24.8 Mb in Duroc. The QTL region is one of the few reported for androstenone that does not simultaneously affect levels of other sex steroids. The main objective of this study was to fine map this QTL. Whole genome sequence data from 23 Norwegian Duroc boars were analyzed to detect new polymorphisms within the QTL region. A subset of 161 SNPs was genotyped in 834 Duroc sires and analyzed for association with androstenone in adipose tissue and testosterone, estrone sulphate and 17β‐estradiol in blood plasma. Our results revealed 100 SNPs significantly associated with androstenone levels in fat (P < 0.001) with 94 of the SNPs being in strong linkage disequilibrium in the region 23.03–24.27 Mb. This haplotype block contains at least four positional candidate genes (HSD17B6, SDR9C7, RDH16 and STAT6) involved in androstenone biosynthesis. No significant associations were found between any of the SNPs and levels of testosterone and estrogens, confirming previous findings. The amount of phenotypic variance explained by single SNPs within the haplotype block was as high as 5.4%. As the SNPs in this region significantly affect levels of androstenone without affecting levels of other sex steroids, they are especially interesting as genetic markers for selection against boar taint.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.     

Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline-sulfonamide as hybrid antiprotozoal agents

A new series of 4-aminochloroquinoline based sulfonamides were synthesized and evaluated for antiamoebic and antimalarial activities. Out of the eleven compounds evaluated (F1–F11), two of them (F3 and F10) showed good activity against Entamoeba histolytica (IC₅₀ <5 μM). Three of the compounds (F5, F7 and F8) also displayed antimalarial activity against the chloroquine-resistant (FCR-3) strain of Plasmodium falciparum with IC₅₀ values of 2 μM. Compound F7, whose crystal structure was also determined, inhibited β-haematin formation more potently than quinine. To further understand the action of hybrid molecules F7 and F8, molecular docking was carried out against the homology model of P. falciparum enzyme dihydropteroate synthase (PfDHPS). The complexes showed that the inhibitors place themselves nicely into the active site of the enzyme and exhibit interaction energy which is in accordance with our activity profile data. Application of Lipinski ‘rule of five’ on all the compounds (F1–F11) suggested high drug likeness of F7 and F8, similar to quinine.