The effect of 5'-(N,N-dimethyl)-amiloride on cytotoxic activity of doxorubicin and vincristine in CEM cell lines

Intracellular pH (pHi) plays an important role in anticancer drug accumulation in cancer cells. Resistant cells often express membrane P-glycoprotein responsible for active drug extrusion and participating in increased pHi. In the present paper, we report on the influence of Na+/H+-exchanger inhibitor, 5'-(N,N-dimethyl)-amiloride (AMI), on the cytotoxic effects of doxorubicin (DOXO) and vincristine (VCR) in the parental CEM, and resistant CEM/DNR and CEM/VCR cell lines. The obtained results revealed a potentiating effect of AMI to both anticancer drugs in parental CEM line. However, AMI did not significantly potentiate the effect of DOXO or VCR in resistant CEM cell lines. We conclude, that inhibition of Na+/H+-exchanger by AMI is not sufficient for reversal of drug resistance in the tested CEM/DNR and CEM/VCR cell lines and the possible change in pHi does not affect the mechanisms of cell resistance.     

5-(N,N-Dimethyl)-amiloride to discriminate the Unidirectional electrolyte transports in rat small intestine and proximal colon in vivo

The effect of dimethyl-amiloride (DMA), a selective Na+/H+ exchange blocker, was studied on electrolyte net fluxes and unidirectional fluxes of Na and Cl at four levels of rat intestine in vivo in basal conditions. DMA was applied intraluminally at concentrations of 10(-4) and 10(-3) M in the model of ligated loops prepared from duodenum, proximal jejunum, distal ileum and ascending colon in fasted Sprague Dawley rats. Two iso-osmotic test solutions were used: (1) hypo-ionic: Na+ 80 mM and (2) iso-ionic: Na+ 148 mM, pH 8.2. 22Na was placed in the loop and 36Cl was given by intravenous route at the beginning of the experiment. Na+/H+ was calculated by two different means, one was based on pH variation following amiloride inhibition of Na influx, the other on the calculation of the passive Na transport. The quantitative evaluation shows that Na/H exchange largely contributes to the electroneutral absorption and luminal pH regulation. The exchanger activity decreases from duodenum, jejunum, ileum and colon where it is completed by K/H exchange to assure low colon luminal pH.     

Role of Rab5 in insulin receptor-mediated endocytosis and signaling

Activated insulin receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between insulin receptor signaling and endocytosis is not well understood. This study utilizes both overexpression and depletion of Rab5 proteins to show that they play a critical role in both insulin-stimulated fluid phase and receptor-mediated endocytosis. Specifically, Rab5:WT and Rab5:Q79L (a GTP-hydrolysis defective mutant) enhance both types of endocytosis in response to insulin, while Rab5:S34N (a GTP-binding defective mutant) has the opposite effect. Morphological analysis indicates that both Rab5 and insulin receptor are found on early endosomes, but not at the plasma membrane. In addition, expression of Rab5:WT and Rab5:Q79L enhance both Erk1/2 and Akt activation without affecting JN- and p38-kinase activities, while the expression of Rab5:S34N blocks both Erk1/2 and Akt activation. Consistent with these observations, DNA synthesis is also altered by the expression of Rab5:S34N. Taken together, these results demonstrate that Rab5 is required for insulin receptor membrane trafficking and signaling.     

Richard G.W. Anderson (1940-2011) and the birth of receptor-mediated endocytosis

On March 19, 2011, the discipline of cell biology lost a creative force with the passing of Richard G.W. Anderson, Professor and Chairman of the Department of Cell Biology at the University of Texas Southwestern Medical School. An unabashed chauvinist for cell biology, Dick served for many years on the editorial board of The Journal of Cell Biology and the Council of the American Society for Cell Biology. He died of glioblastoma multiforme six days before his 71st birthday.     

Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes.

In cultured human fibroblasts we observed that monensin, a Na+/H+-exchanging ionophore, (i) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme, (ii) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of beta-hexosaminidase, (iii) induces secretion of mature beta-hexosaminidase and mature cathepsin D, and (iv) inhibits carbohydrate processing in and proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium, about half of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi apparatus [Tartakoff (1980) Int. Rev. Exp. Pathol. 22, 227-250] may be the cause of some of the effects observed in the present study (iv). Other effects (i, ii) are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.     

Structural characterization and kinetics of nitric-oxide synthase inhibition by novel N5-(iminoalkyl)- and N5-(iminoalkenyl)-ornithines

Isoform-specific nitric-oxide synthase (NOS) inhibitors may prove clinically useful in reducing the pathophysiological effects associated with increased neuronal NOS (nNOS) or inducible NOS (iNOS) activity in a variety of neurological and inflammatory disorders. Analogs of the NOS substrate L-arginine are pharmacologically attractive inhibitors because of their stability, reliable cell uptake, and good selectivity for NOS over other heme proteins. Some inhibitory arginine analogs show significant isoform selectivity although the structural or mechanistic basis of such selectivity is generally poorly understood. In the present studies, we determined by x-ray crystallography the binding interactions between rat nNOS and N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO), a previously identified mechanism-based, irreversible inactivator with moderate nNOS selectivity. We have also synthesized and mechanistically characterized several L-VNIO analogs and find, surprisingly, that even relatively minor structural changes produce inhibitors that are either iNOS-selective or non-selective. Furthermore, derivatives having a methyl group added to the butenyl moiety of L-VNIO and L-VNIO derivatives that are analogs of homoarginine rather than arginine display slow-on, slow-off kinetics rather than irreversible inactivation. These results elucidate some of the structural requirements for isoform-selective inhibition by L-VNIO and its related alkyl- and alkenyl-imino ornithine and lysine derivatives and may provide information useful in the ongoing rational design of isoform-selective inhibitors.     

Enzymatic synthesis and characterization of N5-(carboxymethyl)-L-ornithine and N6-(carboxymethyl)-L-lysine

Summary This report describes the enzyme-catalyzed synthesis, characterization, and chromatographic separation of N⁶-(carboxymethyl)-L-lysine and N⁵-(carboxymethyl)-L-ornithine. The two N -(carboxyalkyl)amino acids are formed via a reductive condensation between glyoxylate and the- or-amino groups of lysine and ornithine, respectively. Both reactions are catalyzed by the NADPH-dependent enzyme, N⁵-(carboxyethyl)ornithine synthase [EC 1.5.1.24], found in some strains of the lactic acid bacteriumLactococcus lactis subsp.lactis.     

Effect of acidic extracellular pH on the receptor-mediated endocytosis of peroxidase-antiperoxidase (PAP) immune complex in rat peritoneal macrophages

The effect of acidic extracellular medium on the uptake of peroxidase-antiperoxidase (PAP) immune complex in rat peritoneal macrophages was studied with the electron microscope. The acidic extracellular medium resulted in acidification of the cytosol in a relatively short period of time as was shown with 5(6) carboxyfluorescein as indicator. At pH 5.5 PAP was internalized. Some of the ligand was found in endosomes; however, a considerable quantity was still present at the cell surface after 5 min internalization. By 30 min the surface was cleared of PAP and the ligand was detectable almost exclusively in endosomes. PAP was never found in lysosomes which were identified by previous loading with cationized ferritin. We conclude that a pH shift of the cytoplasm results in inhibition of endosome-lysosome fusion.     

Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis

Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast alpha-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on alpha-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.     

Discovery of betrixaban (PRT054021), N-(5-chloropyridin-2-yl)-2-(4-(N,N-dimethylcarbamimidoyl)benzamido)-5-methoxybenzamide,a highly potent,selective, and orally efficacious factor Xa inhibitor

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.     

Mechanism of formation of 5-(hydroxymethyl)-2-furaldehyde from D-fructose an sucrose

The literature contains two alternative hypotheses for the mechanism of dehydration of fructose to 5-(hydroxymethyl)-2-furaldehyde (HMF), namely (1) a sequence of reactions commencing with and retaining the fructofuranose ring intact, and (2) a succession of reactions proceeding mainly via open-chain intermediates. The existing evidence for hypotheses (1) and (2) is reviewed and found to favor (1). The major products from fructose in water at 250 degrees, (with and without acid catalysis) have been investigated on a time-resolved basis and analysis of the results was found to confirm the first hypothesis. A necessary fructofuranosyl-cation intermediate in this hypothesis is produced directly by the hydrolysis of sucrose, and reacts to produce HMF in high yields.     

Effects of prolonged exposure to ammonia on fluid-phase,receptor-mediated,and adsorptive (non specific) endocytosis in cultured neuroblastoma cells

Summary The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells

By using Western blot and RT-PCR analyses, the expression of ClC-5, a member of the ClC family of voltage-gated chloride channels, and its mRNA was detected in OK cells. The effect of chloride channel inhibitors on receptor-mediated endocytosis of albumin was examined in OK cells and compared to that of vacuolar H(+)-ATPase inhibitors. Accumulation of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a chloride channel inhibitor, in a concentration-dependent fashion. In contrast, uptake of FITC-inulin, a fluid-phase endocytosis marker, was not affected by NPPB. Other chloride channel inhibitors, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid and diphenylamine-2-carboxylic acid, also inhibited FITC-albumin uptake. NPPB, as well as a vacuolar H(+)-ATPase inhibitor bafilomycin A(1), caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. These results suggest that chloride channel, most likely ClC-5, plays an important role in the receptor-mediated endocytosis of albumin in OK cells.     

受体介导内吞对巨噬细胞膜电位、胞浆和溶酶体pH的影响

本文利用荧光标记方法测定了刀豆素Ａ、麦芽凝集素、酵母多糖刺激引起的巨噬细胞膜电位、胞浆ｐＨ溶酶体ｐＨ的变化。结果显示三种配体均导致细胞膜电位超极化,胞浆ｐＨ降低、溶酶体ｐＨ或高,三个生理参量趋于稳定时间稍有不同。胞浆ｐＨ的降低可能有抑制内吞的作用,溶酶体ｐＨ上升是触发溶酶体内容物外排的基本因素。内吞引起的这些变化是细胞代谢过程中自我调节和保护的表现。     

Studies on N4-(2-deoxy-D-pentofuranosyl)-4,6-diamino-5-formamidopyrimidine (Fapy.dA) and N6-(2-deoxy-D-pentofuranosyl)-6-diamino-5-formamido-4-hydroxypyrimidine (Fapy.dG).

Exposure of DNA to oxidative stress produces a variety of DNA lesions including the formamidopyrimidines, which are derived from the purines. These lesions may play important roles in carcinogenesis. We achieved the first chemical syntheses of a monomeric form of Fapy-dA (1) and oligonucleotides containing this lesion or Fapy-dG at a defined site. Monomeric Fapy-dA readily epimerized at 25 degrees C in phosphate buffer (pH 7.5). The beta-anomer was favored by a ratio of 1.33:1.0, and equilibration was achieved in less than 7 h. Deglycosylation of Fapy-dA in the monomer follows first-order kinetics from 37 to 90 degrees C. The rate constants for deglycosylation of Fapy-dA in the monomeric and oligonucleotide substrates were measured at a common temperature (55 degrees C) and found to be the same within experimental error (t(1/2) = 20.5 h). Implementation of the activation parameters measured for the deglycosylation of 1 indicates that the half-life for deglycosylation of Fapy-dA at 37 degrees C is approximately 103 h. Analysis of the rate constant for deglycosylation of Fapy-dG in an oligonucleotide, revealed that this lesion is approximately 25 times more resistant to hydrolysis than Fapy-dA at 55 degrees C. These results indicate that Fapy-dA and Fapy-dG will be sufficiently long-lived in DNA so as to warrant investigation of their genotoxicity, and both anomers will be present during this time.     

Inhibitory Effect of 8-(N,N-Diethylamino)octyl-3, 4, 5-Trimethoxybenzoate Hydrochloride (TMB-8) on Germination of Bacillus cereus T Spores

Effect of 8-(N,N-diethylamino)octyl-3, 4, 5 - trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L -alanine and inosine was investigated. TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores. The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L -alanine. Addition of Ca²⁺ reversed the inhibitory effect of TMB-8 in a dose-related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.     

Wheat germ agglutinin uptake by cytotoxic lymphocytes,a quantitative model of receptor-mediated endocytosis

Summary The binding and uptake of fluorescence labeled wheat germ agglutinin into cytotoxic T-cells was measured by single cell cytophotometric analysis. The intensity of fluorescence in these cells increased continuously over 24 hrs, indicating a permanent turnover of the ligands for WGA. Although the labeling of the cells was intense, no change in the proliferation rate of this interleukin-2 dependent cell line was observed. Therefore no interaction between the interleukin-2 receptor and other receptors regulating the cellular proliferation with the lectin is likely.Abbreviations au arbitary units - CTLL-1 murine cytotoxic interleukin-2 dependent cell line - FCS fetal calf serum - FITC fluoresceinisothiocyanate - HEPES hydroxyethylpiperazine-ethanesulfonic acid - MHC major histocompatibility complex - WGA wheat germ agglutinin