Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

In vitro effects of arachidonic acid on the prostaglandin synthesizing system in gastric mucosa

Self-destruction of the prostaglandin cyclooxygenase has been suggested to be an important factor in the regulation of endogenous prostaglandin synthesis. The present study was done in order to define the role of this substrate-induced inactivation in the regulation of prostaglandin synthesis in gastric mucosa. In tissue homogenate, the prostaglandin synthesizing capacity is rapidly inactivated at 37 degrees C, even in the absence of exogenous arachidonic acid. It was shown that this inactivation can be prevented both by EDTA as a chelator of calcium-ions and by tetracaine, a specific inhibitor of the phospholipase A2. Additional exogenous arachidonic acid again inactivated prostaglandin synthesis in a dose dependent manner. In contrast, the prostaglandin synthesizing capacity in organ cultured mucosal biopsies is well preserved, although the release of endogenous substrate was activated by extracellular calcium and Ca-ionophore A23187. Furthermore, even at high concentrations of exogenous arachidonic acid present in the culture medium, the synthesizing capacity in intact biopsies was only slightly and reversibly reduced. These large differences between intact biopsies and cell free tissue preparations point to very efficient mechanisms controlling the substrate availability for the cyclooxygenase system both from endogenous and exogenous sources in intact gastric mucosa.     

Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

In a previous report we documented an increased Na⁺-dependent transport of inorganic phosphate (P _i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P _i -uptake stimulator) lead to a 3-4-fold stimulation of Na⁺-dependent P _i -uptake (10ng cRNA injected, 3–5 days of expression). Na⁺-independent uptake of P _i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K _m -values for the induced Na⁺-dependent uptake were 0.26 ± 0.04 mm for P _i and 14.8 ± 3.0 mm for Na⁺. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P _i -uptake into Xenopus laevis oocytes, but which is not a P _i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996     

Evaluation by electron microscopy of the integrity of iodinated plasmalipoproteins

Summary Iodination of proteins and lipoproteins is a widely used in vitro labelling procedure in metabolic, autoradiographic and various other studies. However, all available iodination techniques have involved the possible damage to the proteins by self-irradiation, oxidizing agents, the alkaline milieu or by the introduction of iodine into the molecular structure itself. To evaluate the integrity of iodinated lipoproteins, we observed the electron microscopic appearance of normal and iodinated rabbit very low density lipoproteins (VLDL) by negative staining with phosphotungstic acid. Iodination up to a molar iodine/protein ratio of 2.89 did not result in any change of shape, size or aggregating tendency of the particles. No stacks or disk-like particles like those of various hyperlipoproteinemic states were found. We conclude that electron microscopy is a valuable tool in assessing the morphological appearance of lipoprotein iodination, but it should be complemented by other techniques.     

Selenium content of livers from sex-linked dwarf and normal broiler breeders

Plasma concentrations of cGH, T₃, and T₄ were not different between dwarf and normal broiler breeders. Normal hens had a liver selenium content of 710±35 ng/g, and dwarf hens 656 ±nine ng/g (n=8). Following injections into a wing vein of different doses (1.5, 3, 6, 12, and 24 μg/kg) of the hypothalamic hormone TRH, GH was increased after 15 min. This effect seemed to last longer in dwarf chickens. Plasma concentrations of T₃ increased significantly 1 h after TRH in normal hens, but TRH was ineffective in raising T₃ levels in dwarf animals. The selenium content of livers obtained following decapitation after 2 h was also increased in normal hens up to 902±42 ng/g using the highest dose of TRH (24 μg/kg). This seemed not to be the case for dwarf animals. A much smaller. number of hepatic cGH receptors was also found in dwarf hens, whereas the affinity of the hepatic GH receptor was not influenced by the genotype. It is concluded that the sex-linked dwarf hens are unable to increase their hepatic T₄ into T₃ conversion following a TRH challenge probably because of a deficiency in hepatic GH receptors. The lower content of selenium in dwarfs and their inability to increase its uptake after TRH seem therefore to support the hypothesis that selenium has a direct role in the activity of the 5′-deiodinase complex.     

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac? and 2000 for Spectrum? library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.     

Improving the phenotype predictions of a yeast genome‐scale metabolic model by incorporating enzymatic constraints

Genome‐scale metabolic models (GEMs) are widely used to calculate metabolic phenotypes. They rely on defining a set of constraints, the most common of which is that the production of metabolites and/or growth are limited by the carbon source uptake rate. However, enzyme abundances and kinetics, which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme's abundance and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between and within metabolic pathways. The developed method and model are expected to increase the use of model‐based design in metabolic engineering.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.