A highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

Total antioxidant capacity – a novel early bio-chemical marker of oxidative stress in HIV infected individuals

Background  

Oxidative stress induced by the production of reactive oxygen species may play a critical role in the stimulation of HIV replication and the development of immunodeficiency. This study was conducted as there are limited and inconclusive studies on the significance of a novel early marker of oxidative stress which can reflect the total antioxidant capacity in HIV patients,     

Diets high in selenium and isoflavones decrease androgen-regulated gene expression in healthy rat dorsolateral prostate

Background  

High dietary intake of selenium or soybean isoflavones reduces prostate cancer risk. These components each affect androgen-regulated gene expression. The objective of this work was to determine the combined effects of selenium and isoflavones on androgen-regulated gene expression in rat prostate.     

“On A Piece of Chalk”-Updated1

ABSTRACT. Many advances have been made in our knowledge of the biology of foraminifera over the past several decades. Fine structural, biophysical, and molecular biological studies have shown that the most prominent components of their distinctive bidirectional granuloreliculopods are bundles of micro tubules linked by crossbridges to each other, as well as to membrane-bound organelles and the plasma membrane. the microtubules ratchet past each other as dynein transduces the free energy of ATP to produce pseudopodal movements. In spite of the fact that there are over 40,000 described species of living and fossil species of foraminifera, there have been many recent exciting discoveries of new species and groups. New casting techniques are providing us with greater understanding of the complexities and functional aspects of form in the group. Significant advances are being made in understanding the distribution and energetics of deep-sea forms. Larger and planktonic foraminifera are the hosts for a particularly diverse range of endosymbiotic algae, including dinoflagellates, chlorophytes, unicellular rhodophytes, and diatoms. Chloroplast husbandry also occurs. Significant research effort has been expended yielding us considerable insight into various aspects of the endosymbiotic phenomenon. A unified conceptual framework has been drawn to help us understand the life cycle options found in foraminifera.     

Influences of dietary soy isoflavones on metabolism but not nociception and stress hormone responses in ovariectomized female rats

Background  

Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. Few studies have examined the nociception and stress hormone responses after consumption of soy isoflavones.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

A stem-loop "kissing" model for the initiation of recombination and the origin of introns

Mutations which improve the efficiency of recombination should affect either the proteins which mediate recombination or their substrate, DNA itself. The former mutations would be localized to a few sites. The latter would be dispersed. Studies of hybridization between RNA molecules have suggested that recombination may be initiated by a homology search involving the "kissing" of the tips of stem loops. This predicts that, in the absence of other constraints, mutations which assist the formation of stem loops would be favored. From comparisons of the folding of normal and shuffled DNA sequences, I present evidence for an evolutionary selection pressure to distribute stem loops generally throughout genomes. I propose that this early pressure came into conflict with later local pressures to impose information concerning specific function. The conflict was accommodated by permitting sections of DNA concerned with a specific function to evolve in dispersed segments. Traces of the conflict seem to be present in some modern intron-containing genes. Thus, introns may have allowed the interspersing of selectively advantageous stem loops in coding regions of DNA.      

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.