UAP1 is overexpressed in prostate cancer and is protective against inhibitors of N-linked glycosylation.

Prostate cancer is the second most common cause of cancer-associated deaths in men, and signaling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Consequently, AR target genes are prominent candidates to be specific for prostate cancer and also important for the survival of the cancer cells. Here we assess the levels of all hexosamine biosynthetic pathway (HBP) enzymes in 15 separate clinical gene expression data sets and identify the last enzyme in the pathway, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), to be highly overexpressed in prostate cancer. We analyzed 3261 prostate cancers on a tissue microarray and found that UAP1 staining correlates negatively with Gleason score (P=0.0039) and positively with high AR expression (P<0.0001). Cells with high UAP1 expression have 10-fold increased levels of the HBP end-product, UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is essential for N-linked glycosylation occurring in the endoplasmic reticulum (ER) and high UAP1 expression associates with resistance against inhibitors of N-linked glycosylation (tunicamycin and 2-deoxyglucose) but not with a general ER stress-inducing agent, the calcium ionophore A23187. Knockdown of UAP1 expression re-sensitized cells towards inhibitors of N-linked glycosylation, as measured by proliferation and activation of ER stress markers. Taken together, we have identified an enzyme, UAP1, which is highly overexpressed in prostate cancer and protects cancer cells from ER stress conferring a growth advantage.

Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes.

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

RNA aptamers to S-adenosylhomocysteine: kinetic properties, divalent cation dependency, and comparison with anti-S-adenosylhomocysteine antibody.

To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 10(14) variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the "classic" ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.

EGF-induced activation of Stat1, Stat3, and Stat5b is unrelated to the stimulation of DNA synthesis in cultured hepatocytes.

Transcription factors of the STAT family have been implicated in regulation of cell proliferation. EGF activates several STAT proteins in liver. We have studied the relationship between STAT activation and the growth-stimulatory effect of EGF in rat hepatocytes, assessing specific DNA-binding activity of STAT proteins in electrophoretic mobility-shift and supershift assays. In freshly isolated hepatocytes, EGF activated Stat1, Stat3, and, particularly, Stat5b. However, the ability of EGF to produce this activation was rapidly attenuated when the cells were cultured, while the activation by IFN-gamma (Stat1) and IL-6 (Stat3) was sustained. Hepatocytes cultured for 24-48 h are highly sensitive to the stimulatory effect of EGF on S phase entry. In these cells EGF did not detectably activate Stat1, Stat3, or Stat5b but markedly stimulated MAP kinase (Erk1/2). Thus, although EGF has the ability to activate several STAT proteins, this did not seem to be part of the mitogenic mechanisms used by the EGF receptor in hepatocytes.

Adhesive peptides selected by phage display: characterization, applications and similarities with fibrinogen.

Phase clones with affinity for polystyrene/polyurethane magnetic particles were isolated from a 10-men peptide display library. Sequence analysis revealed that 40 out of 80 clones contained the consensus WXXWXXXW. Some of the selected phages showed high surface activity and adsorbed to plastic surfaces even in the presence of blocking agents or surfactants. Covalent attachment of a synthetic peptide (KG), carrying one of the selected sequences to alkaline phosphatase (AP) or bovine serum albumin (BSA) enhanced binding of AP to a wide range of materials and improved the ability of BSA to prevent binding of antibodies and phages to polystyrene. Interestingly, the WXXW/XXXW motif occurs in the beta- and gamma-chains of the natural "adhesive" protein fibrinogen, and a synthetic peptide carrying the gamma-chain 369-376 sequence turned out to have essentially the same binding properties as the KG peptide. Furthermore, adsorption in different types of polystyrene was similar for AP carrying either the KG or gamma-chain peptide intact fibrinogen and plasmin-generated fragment D1. The latter fragment contains two copies of the WXXWXXXW motif but lacks the alpha-chain: protuberances previously implicated in fibrinogen adsorption. Thus, our study may have revealed a hitherto unknown structural determinant for fibrinogen's adsorptivity, located in the 13-kDa C terminal region of the gamma-chain.

Colocalization of gamma-aminobutyrate and gastrin in the rat antrum: an immunocytochemical and in situ hybridization study.

BACKGROUND/AIMS: The inhibitory neurotransmitter gamma-aminobutyrate (GABA) has been shown to coexist with insulin in pancreatic beta-cells. We have presently investigated whether GABA also colocalizes with gastrin in G cells in rat antral mucosa. METHODS: Three alternative approaches were used: (1) gastrin in situ hybridization and GABA immunocytochemistry on consecutive cryostat sections; (2) GABA immunocytochemistry and gastrin immunocytochemistry on adjacent semithin and ultrathin sections; and (3) double-immunogold labeling of GABA and gastrin in the same ultrathin section. RESULTS: Colocalization of GABA and gastrin was observed with each of the three approaches. In the double-immunogold labeled cells, the G-cell granules displayed a high gold-particle density indicating gastrin and a low particle density indicating GABA, whereas the converse was true for the extragranular cytoplasmic matrix. The gold-particle ratios between these compartments were 11 (for gastrin) and 0.36 (for GABA), respectively. GABA labeling was also observed in two other antral endocrine cell types, classified by morphological criteria as somatostatin producing D cells and serotonin producing ECn cells. CONCLUSIONS: This is the first direct demonstration of GABA in gastrointestinal G cells. Our findings suggest that GABA may have a paracrine function in the stomach mucosa, analogous to its presumed role in the pancreatic islets.

Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study.

The distributions of the mRNAs encoding the L-glutamate transporters GLT1 and GLAST were examined in the rat brain by in situ hybridization using 35S-labelled oligonucleotide probes. Probes directed to GLT1 produced dense labelling in the hippocampus, neocortex and neostriatum, and weak labelling in the cerebellum. In contrast, GLAST mRNA appeared to be greatly enriched in the cerebellum compared to other brain regions. While the intensity of the labelling for GLAST and GLT1 varied among different regions, their cellular distributions appeared to coincide inasmuch as both mRNAs were mainly expressed by glial cells. Labelling occurred, inter alia, in glial cells throughout the hippocampus, and in Golgi epithelial cells in the Purkinje cell layer of the cerebellum.