Identification of gastric cancer patients by serum protein profiling

Using surface-enhanced laser desorption ionization mass spectrometry (SELDI/TOF-MS) and ProteinChip technology, coupled with a pattern-matching algorithm and serum samples, we screened for protein patterns to differentiate gastric cancer patients from noncancer patients. A classifier ensemble, consisting of 50 decision trees, correctly classified all gastric cancers and all controls of a training set (100% sensitivity and 100% specificity). Eight of 9 stage I gastric cancers (88.9% sensitivity for stage I) were correctly classified. In addition, 28 sera from gastric cancer patients taken in different hospitals were correctly classified (100% sensitivity). Furthermore, all 11 control sera obtained from patients without gastric cancer (100% specificity) were classified correctly and 29 of 30 healthy blood-donors were classified as noncancerous. ProteinChip technology in conjunction with bioinformatics allows the highly sensitive and specific recognition of gastric cancer patients.     

Radioresistance-related proteins in rectal cancer

Radiation therapy (RT), combined with chemotherapy, is currently the standard adjuvant approach for UICC-stage II and III rectal cancer patients. Individual rectal tumors display wide ranges of radiosensitivity (RS). The aim of the present study was to identify proteins associated with radioresistance (RR), with the final aim of predicting tumor response. Seventeen patients were recruited between July 1998 and November 2001. All patients suffered from a locally advanced adenocarcinoma of the rectum. Tumor biopsies were taken before RT. All patients received preoperatively 50 Gray or a biologically equivalent total dose. Surgery was performed after 6 weeks, and response assessed histopathologically. Seven tumors showed complete response, seven a partial response, and in three patients only microscopic disease remained. Proteins were separated using narrow pH gradient two-dimensional polyacrylamide gel electrophoresis and silver staining. Automatic gel comparison allowed matching a mean number of 497 +/- 280 spots. Forty-four spots showing significant differential expression in RR tumors were localized on the pH 4.5-5.5 gels, 33 out of them being identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Twenty-two out of 37 spots of interest could be identified on the pH 5.5-6.7 gels. The expression of the following proteins correlates with RR: tropomodulin (p = 0.01), heat shock protein 42 (p = 0.03), beta-tubulin (p = 0.10), annexin V (p = 0.10), calsenilin (p = 0.10), or with radiosensitivity (RS): keratin type I (p = 0.03), notch 2 protein homolog (p = 0.05) and DNA repair protein RAD51L3 (p = 0.11). A further RR-related protein is so far only hypothetical: XP_030188 (NCBI Database accession number, p = 0.14). We consider the fact that several of these proteins of interest are known to be associated with RR as a valid proof of principle for this proteomics approach. These results will serve as a basis for developing an assay for testing rectal cancers for radioresistance.     

Enzyme assays for high-throughput screening

Assaying enzyme-catalyzed transformations in high-throughput is crucial to enzyme discovery, enzyme engineering and the drug discovery process. In enzyme assays, catalytic activity is detected using labelled substrates or indirect sensor systems that produce a detectable spectroscopic signal upon reaction. Recent advances in the development of high-throughput enzyme assays have identified new labels and chromophores to detect a wide range of enzymes activities. Enzyme activity profiling and fingerprinting have also been used as tools for identification and classification, while microarray formats have been devised to increase throughput.     

Spatial distributions of expansion rate, cell division rate and cell size in maize leaves: a synthesis of the effects of soil water status, evaporative demand and temperature

The spatial distributions of leaf expansion rate, cell division rate and cell size was examined under contrasting soil water conditions, evaporative demands and temperatures in a series of experiments carried out in either constant or naturally fluctuating conditions. They were examined in the epidermis and all leaf tissues. (1) Meristem temperature affected relative elongation rate by a constant ratio at all positions in the leaf. If expressed per unit thermal time, the distribution of relative expansion rate was independent of temperature and was similar in all experiments with low evaporative demand and no water deficit. This provides a reference distribution, characteristic of the studied genotype, to which any distribution in stressed plants can be compared. (2) Evaporative demand and soil water deficit affected independently the distribution of relative elongation rate and had near-additive effects. For a given stress, a nearly constant difference was observed, at all positions of the leaf, between the relative elongation rates of stressed plants and those of control plants. This caused a reduction in the length of the zone with tissue elongation. (3) Methods for calculating cell division rate in the epidermis and in all leaf tissues are proposed and discussed. In control plants, the zone with cell division was 30 mm and 60 mm long in the epidermis and in whole tissues, respectively. Both this length and relative division rate were reduced by soil water deficit. The size of epidermal and of mesophyll cells was nearly unaffected in the leaf zone with both cell division and tissue expansion, suggesting that water deficit affects tissue expansion rate and cell division rate to the same extent. Conversely, cell size of epidermis and mesophyll were reduced by water deficit in mature parts of the leaf.     

Emergence of young human genes after a burst of retroposition in primates

The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past ∼63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome−derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.     

HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells

Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ-restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide.     

Prominent role of the Ig-like V domain in trans-interactions of nectins. Nectin3 and nectin 4 bind to the predicted C-C'-C"-D beta-strands of the nectin1 V domain

Nectins form a family of integral molecules that belong to the immunoglobulin superfamily. Their ectodomain is made of three Ig-like domains (V, C, C). This family comprises at least five members, namely nectin1, -2, -3, -4, and poliovirus receptor (PVR), that are involved in different physiological and pathological processes. (i) Nectins are adhesion molecules localized at adherens junctions in epithelial cells. (ii) Some nectins act as poliovirus or alpha-herpesvirus receptors (nectin1). (iii) Nectin1 mutations are involved in orofacial developmental abnormalities in humans. Adhesion properties of nectins are mediated by Ca(2+)-independent homophilic and heterophilic processes through ectodomain trans-interactions. We have described a nectin trans-hetero-interaction network: nectin3 binds to nectin1, nectin2, and PVR; nectin1 also binds to nectin4. In the present study we compared the affinities of the different trans-interactions mediated by nectin1. We found that the K(D) of nectin1/nectin3 and nectin1/nectin4 interactions is 1 and 100 nm, respectively, whereas the K(D) of the nectin1-mediated homophilic interaction is 1 microm. We show that nectin1/nectin3 and nectin1/nectin4 trans-hetero-interactions were mediated through trans V to V domain interactions, whereas C domains contributed to increase the affinity of the interaction. Nectin3 and nectin4 share a common binding region in the nectin1 V domain: (i) nectin3 strongly competed with nectin4 binding, (ii) nectin3 and nectin4 binding to nectin1 was reduced by a number of monoclonal antibodies directed against the nectin1 V domain, and (iii) the glycoprotein D of herpes simplex virus-1 that binds to the V domain of nectin1 reduced nectin3 and nectin4 binding. Finally, using chimeric nectin1/PVR receptors where PVR V domain beta-strands were substituted with the corresponding regions of nectin1, the nectin3 and nectin4 minimal binding region on nectin1 V domain was mapped to the C-C'-C"-D beta-strands.     

Les Stephanodiscus (Diatomées) du Lac Léman: Description d'une nouvelle espèce,Stephanodiscus irregularis; mise au point taxonomique et distributions saisonnières

Intact sediment-water columns from a flowing cypress swamp receiving primary wastewater effluent were used to evaluate inorganic N removal and to determine the fate of ¹⁵NH _inf4 ^sup+ -N added to the floodwater. Treatments represented wetland sites which had received 0 (initial application), 2, and 50 years of primary wastewater application. The rate of inorganic-N decrease in the floodwater was greatest for the initial application columns, primarily due to sediment adsorption of NH _inf4 ^sup+ , followed by 2-year and 50-yr-columns. Maximum removal rates were 318, 296, and 148 mg N m^–2 day^–1, respectively. At the end of the 21-day study period, only 0.5 to 2.3% of applied ¹⁵N was recovered in the floodwater and 11.4 to 17.3% was recovered in the sediment, with the remaining 82.2 to 86.3% being lost from the sediment-water system. Results of the study indicated that N removal efficiency did not decrease with prolonged wastewater application, despite reduced sediment adsorption capacity, because of the significance of gaseous N losses (nitrification-denitrification, NH₃ volatilization) as an N sink in the sediment-water system.Florida Agricultural Experiment Stations Journal Series No. 7712     

Quantification of abscisic Acid in a single maize root

Quantitative analyses of abscisic acid in the elongating zone of a single maize root (Zea mays L. cv LG 11) were performed by gas chromatography-mass spectrometry using negative chemical ion ionization. Data showed that the more abscisic acid, the slower the growth, but a large dispersion of individual values was observed. We assume that abscisic acid is perhaps not correlated only to the growth rate.     

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.