Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.     

Auxin transport under saline growth conditions

Zusammenfassung Ein erhöhter Salzgehalt des Substrats hemmt das Koleoptilen-Längenwachstum vonZea mays, onne indessen einen Einfluss auf den Auxintransport und dessen Polarität auszuüben.

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I am thankful to the Pakistan and Danish Atomic Energy Commissions for the award of a fellowship under which the above work was carried out, and to my colleagues at Risø for their help and hospitality.     

Estimation of diffusible auxin under saline growth conditions

Zusammenfassung Der Auxingehalt von Koleoptilenspitzen vonZea mays L.-Keimlingen wird durch saline Wachstumsbedingungen herabgesetzt.

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Part of the work supported by a grant from USDA under the PL-480 research programme.     

Oenocyte and prothoracic gland activity inManduca sexta under varying photoperiod and light conditions

Summary Manduca sexta larvae were subjected to diapause-inducing and diapause-preventing photoperiods, using two types of fluorescents (Indorsun and Blacklight-blue). The oenocytes, prothoracic glands (PTG) and ecdysone levels were examined in 3-day-old 5th instar larvae, 2-day-old and 10-day-old pupae. Our results indicate that oenocytes and PTG cells tend to be more active under long photoperiods while oenocytes only are active under short photoperiods in pupae in diapause. UV light has a definite effect on oenocytes while PTG cells seem to be unaffected. Ecdysone and ecdysterone levels vary with PTG and oenocyte activity at the pupal stage. The significance of these findings is discussed.This work was supported by a research associateship to L.M. by the People's Republic of China and a grant from NSERC to B.J.R.P.     

Macropinocytosis,mTORC1 and cellular growth control

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysosomal surface where Rheb directly binds to and activates mTORC1. Rag is activated by both lysosomal luminal and cytosolic amino acids; Rheb activation requires phosphoinositide 3-kinase, Akt, and the tuberous sclerosis complex-1/2. Signals for activation of Rag and Rheb converge at the lysosomal membrane, and several lines of evidence support the idea that growth factor-dependent endocytosis facilitates amino acid transfer into the lysosome leading to the activation of Rag. This review summarizes evidence that growth factor-stimulated macropinocytosis is essential for amino acid-dependent activation of mTORC1, and that increased solute accumulation by macropinocytosis in transformed cells supports unchecked cell growth.     

Acid phosphatases and hemoglobin in normal and G-6-PD deficient erythrocytes incubated with acetylphenylhydrazine under various conditions

Riassunto L'incubazione con GSSG determina nell'emolizzato una modificazione del quadro elettroforetico delle fosfatasi acide eritrocitarie ed una riduzione della loro attività.Modificazioni identiche a carico dell'enzima possono venire indotte anche nel globulo rosso intero di soggetto normale o di enzimopenico per la G-6-PD, provocando una caduta del tasso di GSH mediante acetilfenilidrazina.     

Cell surface adenylate kinase activity regulates the F1-ATPase/P2Y13-mediated HDL endocytosis pathway on human hepatocytes

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F₁-ATPase stimulates the production of extracellular ADP that activates a P2Y₁₃-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase and nucleoside diphosphokinase activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F₁-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation. Received 13 July 2006; received after revision 29 August 2006; accepted 19 September 2006     

Regulatory T cells, mTOR kinase, and metabolic activity

The field that links immunity and metabolism is rapidly expanding. Apparently, non-immunological disorders such as obesity and type 2 diabetes have been linked to immune dysregulation, suggesting that metabolic alterations can be induced by or be a consequence of an altered self-immune tolerance. In this context, a key role is played by signaling systems acting as metabolic “sensors” linking energy/nutritional status to regulatory T (Treg) cell functions. We propose that a dynamic/oscillatory activity of intracellular metabolism, through mTOR modulation, might represent a shift in understanding the molecular mechanisms governing Treg cell tolerance. In particular, the decision between Treg cell proliferation and hyporesponsiveness arises from their ability to probe the extracellular milieu and, modulating the metabolic intracellular signaling, to determine different qualitative and quantitative functional outcomes.     

Identification of rooting depth and measurements of plant root activity in situ and in toto under field conditions using a gamma probe technique

Summary A technique for measuring root activity of agricultural crops in totality in situ under field conditions has been developed for the first time. The method essentially consists of measuring the activity emanating from roots after plant injection of a gamma radiating isotope using a probe. This also helps continuous monitoring of root growth changes over time.     

Progesterone-induced blocking factor differentially regulates trophoblast and tumor invasion by altering matrix metalloproteinase activity

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while—based on our previous data—PIBF might control trophoblast invasion by suppressing proinvasive genes.     

Δ6-Tetrahydrocannabinol-7-oic acid,a urinary Δ6-THC metabolite: Isolation and synthesis

Zusammenfassung 7-Hydroxy- ⁶-tetrahydrocannabinol (7-Hydroxy- ⁶-THC), ein psychotomimetisch aktiver Metabolit des ⁶-THC, wird von Kaninchen in inaktive ⁶-THC-7 Säure metabolisiert. Die Synthese dieser Säure wird beschrieben.

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The work in Jerusalem was supported by the US National Institute of Mental Health and in Stockholm by the Swedish Medical Research Council.     

Metabolism of pyridine and 3-hydroxypyridine under aerobic,denitrifying and sulfate-reducing conditions

Summary The transformation of pyridine and several hydroxypyridines was investigated under different physiological conditions. Pyridine and 2-, 3- and 4-hydroxypyridine were metabolized aerobically within 9 days in a mineral salt medium inoculated with 10% sewage sludge. Under anaerobic conditions in the presence of nitrate or sulfate, pyridine and 3-hydroxypyridine were metabolized after a lag period of 2 months, while no transformation of 2- or 4-hydroxypyridine was detected. Experiments with¹⁴C-labeled-pyridine showed that under denitrifying as well as under sulfate-reducing conditions pyridine was oxidized to carbon dioxide. Disappearance of nitrate and sulfate under anaerobic conditions was 79% and 85%, respectively, of the amount predicted from the stoichiometric equation for the complete transformation of pyridine.     

Inhibitory effect of exogenous histones on cellular growth and DNA synthesis in Chlamydomonas reinhardtii.]

The addition of exogenous histones to synchronous culture of Chlamydomonas reinhardtii, at the beginning of the cell cycle, leads to the death of the cells. The same amount of histones added in the middle of the cycle, only blocks the cell division. The mechanism of this inhibition effect of the histones could involve a block at the level of the chloroplast DNA replication.