The Arf tumor suppressor protein inhibits Miz1 to suppress cell adhesion and induce apoptosis

Oncogenic stress induces expression of the alternate reading frame (Arf) tumor suppressor protein. Arf then stabilizes p53, which leads to cell cycle arrest or apoptosis. The mechanisms that distinguish both outcomes are incompletely understood. In this study, we show that Arf interacts with the Myc-associated zinc finger protein Miz1. Binding of Arf disrupts the interaction of Miz1 with its coactivator, nucleophosmin, induces the sumoylation of Miz1, and facilitates the assembly of a heterochromatic complex that contains Myc and trimethylated H3K9 in addition to Miz1. Arf-dependent assembly of this complex leads to the repression of multiple genes involved in cell adhesion and signal transduction and induces apoptosis. Our data point to a tumor-suppressive pathway that weakens cell–cell and cell–matrix interactions in response to expression of Arf and that may thereby facilitate the elimination of cells harboring an oncogenic mutation.     

Identification of a Survival-independent Metastasis-enhancing Role of Hypoxia-inducible Factor-1α with a Hypoxia-tolerant Tumor Cell Line

During tumor progression, malignant cells must repeatedly survive microenvironmental stress. Hypoxia-inducible factor-1 (HIF-1) signaling has emerged as one major pathway allowing cellular adaptation to stress. Recent findings led to the hypothesis that HIF-1α may enhance the metastatic potential of tumor cells by a survival-independent mechanism. So far it has not been shown that HIF-1α also directly regulates invasive processes during metastasis in addition to conferring a survival advantage to metastasizing tumor cells. In a hypoxia-tolerant tumor cell line (L-CI.5s), which did not rely on HIF-1 signaling for viability in vitro and in vivo, knockdown of Hif-1α reduced invasiveness of the tumor cells in vitro as well as extravasation and secondary infiltration in vivo. Liver metastases associated induction of proinvasive receptor tyrosine kinase Met phosphorylation as well as gelatinolytic activity were Hif-1α-dependent. Indeed, promoter activity of the matrix metalloproteinase-9 (mmp-9) was shown to be Hif-1α-dependent. This study uncovers a new survival-independent biological function of HIF-1α contributing to the efficacy of metastases formation.     

Regulation of the Ca2+ Channel TRPV6 by the Kinases SGK1, PKB/Akt, and PIKfyve

The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca²⁺ channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active ^S422DSGK1, constitutively active ^T308D,S473DPKB, wild-type PIKfyve, and ^S318APIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (I_Ca) resulting from TRPV6-induced Ca²⁺ entry and subsequent activation of Ca²⁺-sensitive endogenous Cl^? channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes I_H was increased by coexpression of ^S422DSGK1 and by ^T308D,S473DPKB. Coexpression of wild-type PIKfyve further increased I_H in TRPV6 + ^S422DSGK1-expressing oocytes but did not significantly modify I_Ca in oocytes expressing TRPV6 alone. ^S318APIKfyve failed to significantly modify I_Ca in the presence and absence of ^S422DSGK1. ^S422DSGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6.     

The classical pathway of melanogenesis is not essential for melanin synthesis in the adult retinal pigment epithelium

Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I–IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.     

Vertical change in dissolved organic carbon and oxygen at the water table region of an aquifer recharged with stormwater: biological uptake or mixing?

Decreases in dissolved organic carbon (DOC) and dissolved oxygen (DO) with increasing depth below the groundwater table are often considered as evidence for aerobic respiration; however, they may reflect mixing of infiltrating water and groundwater. We found that groundwater DOC concentration was on average 0.3 mg C l^?1 higher and DO concentration 1.5 mg O₂ l^?1 lower at recharge sites replenished with stormwater than at reference sites fed by direct infiltration of rain water from the land surface. Groundwater DOC increased and DO decreased with increasing vadose zone thickness (VZT) at both recharge and reference sites. There was no significant interaction between the effects of stormwater infiltration and VZT. Vertical changes in DOC and DO below the groundwater table at recharge sites could account for by simple mixing of infiltrating stormwater and groundwater. Moreover, aquifer sediment respiration (SR) was not significantly higher at recharge sites than at reference sites. However, slow filtration column experiments showed that SR increased significantly with an increasing supply of easily biodegradable DOC. We conclude that the observed reduction in DOC below the groundwater table at recharge sites was essentially due to water mixing rather than biological uptake because of the low biodegradability of the DOC and the short transit time of stormwater in the upper layers of groundwater. Our results highlight the need to distinguish between the effect of hydrological and biological processes on DOC and DO patterns below the groundwater before conclusions are made on the efficiency of groundwater in degrading surface-derived DOC.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures

To investigate the amount and fate of root-derived C and N, often tracer techniques are used, where plants are labelled with isotopes. In the present study, we evaluated the suitability of the cotton wick method for in situ labelling of peas (Pisum sativum L.) and oats (Avena sativa L.) with ¹³C and ¹⁵N simultaneously. With two greenhouse experiments we investigated how the wick method and aqueous urea and sugar solutions at a variety of concentrations affected plant development. In addition, we investigated the distribution of ¹³C and ¹⁵N in plants from column experiments under outdoor conditions. Solution was taken up by the plant from a small vial connected to the stem by a cotton wick which was passed through a hole in the stem of the plants. Generally, solution uptake varied between individual plants and decreased with increasing sugar concentrations. Below-ground, above-ground and total plant dry matter, were not significantly affected by the wick method and the applied solutions. Mixtures of aqueous glucose solutions at 2 to 4% and aqueous urea solutions at 1% are useful carriers of ¹³C and ¹⁵N. However, in the investigated plants isotopes were not homogeneously distributed among plant parts. Above-ground plant biomass was preferentially enriched with ¹³C and ¹⁵N, whereas below-ground plant biomass was generally lower enriched. Moreover, isotope distribution ratio of individual plants varied considerably, independent of plant part or timing of labelling. This must be taken into account when estimating root-derived C and N. Future studies comparing labelling methods need to present the isotope distribution ratios among plant parts to allow a true comparison of the methods and the evaluation of their suitability for estimating rhizodeposition.     

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple‐category naïve Bayes model, trained on the two‐dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase‐3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross‐family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross‐family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.