Detection of bladder cancer using proteomic profiling of urine sediments

We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival.     

DIG--a system for gene annotation and functional discovery

SUMMARY: We describe a database and information discovery system named DIG (Duke Integrated Genomics) designed to facilitate the process of gene annotation and the discovery of functional context. The DIG system collects and organizes gene annotation and functional information, and includes tools that support an understanding of genes in a functional context by providing a framework for integrating and visualizing gene expression, protein interaction and literature-based interaction networks.     

Diurnal Changes in Metabolite Levels and Crassulacean Acid Metabolism in Kalanchoë daigremontiana Leaves

Diurnal changes in levels of selected metabolites associated with glycolysis, the C₃ cycle, C₄-organic acids, and storage carbohydrates were analyzed in active Kalanchoë daigremontiana Crassulacean acid metabolism leaves. Three metabolic transition periods occurred each day. During the first two hours of light, nearly all of the metabolite pools underwent transient changes. Beginning at daylight, stomata opened transiently and closed again within 30 minutes; malate synthesis continued for about 1 hour into the light; C₃ photosynthesis began within 30 minutes; and net quantities of starch and glucan began to accumulate after 2 hours, continuing linearly throughout the rest of the day.     

Far-red Sensitive Dark Processes Essential for Light- and Gibberellin-induced Germination of Lettuce Seed

The action of prolonged far-red on seed germination was studied in Lactuca sativa L. var. Grand Rapids. Exposure of imbibed seeds to 6 hours far-red before the application of gibberellic acid (GA₃) and thiourea completely prevented germination. Using GA₃, this far-red was effective after the sixth hour of imbibition. At 6, 12, and 18 hours of imbibition equal durations of far-red had equal effects. The kinetics of far-red action was investigated: it was found that although far-red for several hours, irrespective of the energy level, was needed for maximum inhibition, shorter durations (15 and 30 mins) were also appreciably effective provided they were followed by several hours darkness before the supply of GA₃. This is taken to indicate the existence of labile product(s) of the action of a far-red sensitive pigment. Evidence is provided for the existence of promotive dark processes controlled by this pigment, which are essential for germination whether triggered by GA₃, thiourea or red-light. A model for the operation of the pigment system is proposed and its role in the germination mechanism of this seed is discussed.     

Dietary polyunsaturated fatty acids (C18:2 omega6 and C18:3 omega3) do not suppress hepatic lipogenesis

Omega 3 polyunsaturated fatty acids are promoted as beneficial in the prevention of metabolic and cardiovascular diseases. In general, dietary omega 3 fatty acids are derived from plant sources as linolenic acid (LNA, C18:3 omega3) the precursor to eicosapentaenoic acid (EPA, C20:5 omega3) and docosahexaenoic acid (DHA, C22:6 omega3). However, it remains unclear if the polyunsaturated fatty acid (PUFA) LNA can provide the same health benefits as the very long chain highly unsaturated fatty acids (HUFA) EPA and DHA generally derived from oily fish. In this study, mice were fed synthetic diets containing lard (low in PUFA and HUFA), canola oil (to supply PUFA), or a mixture of menhaden and arasco (fish and fungal) oils (to supply HUFA) for 8 weeks. The diets were neither high in calories nor fat, which was supplied at 6%. The lard and canola oil diets resulted in high levels of hepatic triglycerides and cholesterol and elevation of lipogenic gene expression. By comparison livers from mice fed the fish/fungal oil diet had low levels of lipid accumulation and more closely resembled livers from mice fed standard laboratory chow. SREBP1c and PPARgamma gene and protein expression were high in livers of animals fed diets containing lard or canola oil compared with fish/fungal oil. Hepatic fatty acid analyses indicated that dietary PUFA were efficiently converted to HUFA regardless of source. Therefore, differences in hepatic lipid levels and gene expression between dietary groups were due to exogenous fatty acid supplied rather than endogenous pools. These results have important implications for understanding the regulation of hepatic lipogenesis by dietary fatty acids.     

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.     

Cellular Localisation of Adenylyl Cyclase: A Post-genome Perspective

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.Invocation: This paper was written to honour one of the founders of chemical neuroanatomy—Professor Miklós Palkovits on his 70th birthday.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.