Direct interaction and cooperative role of tumor suppressor p16 with band 3 (AE1)

By using the C-terminal 112-residue of band 3 to screen the K562 cDNA library, we find that the p16 interacts with band 3, which was confirmed both in yeast and in mammalian cells. Functional experiments show that p16 facilitates the movement of band 3 to plasma membrane with increased anion transport activity in 293t cells. Moreover, expression of endogenous p16 in 293t cells was increased at 24 and 36 h after transfection with band 3. Our findings provide a novel regulation pathway for both band 3 and p16.     

The many faces of calmodulin in cell proliferation,programmed cell death,autophagy, and cancer

Calmodulin (CaM) is a ubiquitous Ca^2 + receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.     

Alanine racemase of alfalfa seedlings (Medicago sativa L.): first evidence for the presence of an amino acid racemase in plants

We demonstrated several kinds of D-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition of L- or D-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of L- and D-alanine. The K(m) and V(max) values of enzyme for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are 12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K(eq) value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far.     

Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation

The delivery and expression of exogenous genes in plant cells have been of particular interest for plant research and biotechnology. Here, we present results demonstrating a simple DNA transfection system in plants. Short arginine-rich intracellular delivery peptide, a protein transduction domain, was capable of delivering plasmid DNA into living plant cells non-covalently. This peptide-mediated DNA delivery conferred several advantages, such as nuclear targeting, non-toxic effect, and ease of preparation without protoplast formulation. Thus, this novel technology shall provide a powerful tool to investigate gene function in vivo, and lay the foundation for the production of transgenic plants in future.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

Oxidation induces ClC-3-dependent anion channels in human leukaemia cells

To test for redox regulation of anion channels in erythroid cells, we exposed K562 cells to oxidants and measured changes in transmembrane Cl(-) currents using patch-clamp, and in intracellular Cl(-) content using the Cl(-) selective dye MQAE. Oxidation with tert-butylhydroperoxide or H(2)O(2) produced a plasma membrane anion permeability with a permselectivity of NO(3)(-)>lactate(-)>gluconate(-). The permeability increase was paralleled by insertion of ClC-3 protein into the plasma membrane as evident from immunofluorescence microscopy and surface biotinylation. Down-regulation of ClC-3 protein by RNA interference as assessed by immunoblotting decreased the oxidation-stimulated permeability. In conclusion, oxidation induces surface expression of ClC-3 and activation of a ClC-3-dependent anion permeability in K562 cells.     

Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector

The osteoclast variant of the vacuolar H⁺-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC₅₀ in the 10-nanomolar range.     

Cannabinoid receptor ligands mediate growth inhibition and cell death in mantle cell lymphoma

We have earlier reported overexpression of the central and peripheral cannabinoid receptors CB1 and CB2 in mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma. In this study, treatment with cannabinoid receptor ligands caused a decrease in viability of MCL cells, while control cells lacking CB1 were not affected. Interestingly, equipotent doses of the CB1 antagonist SR141716A and the CB1/CB2 agonist anandamide inflicted additive negative effects on viability. Moreover, treatment with the CB1/CB2 agonist Win-55,212-2 caused a decrease in long-term growth of MCL cells in culture. Induction of apoptosis, as measured by FACS/Annexin V-FITC, contributed to the growth suppressive effect of Win-55,212-2. Our data suggest that cannabinoid receptors may be considered as potential therapeutic targets in MCL.     

The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles

A fluorescence method for the direct measurement of Cl^- transport in isolated tonoplast vesicles is described. This technique utilises the Cl^--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl^-, Br^-, I^-, SCN^-, NO ₂ ^- and tetraphenylborate but not by NO ₃ ^- , SO ₄ ^2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl^- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl^- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl^- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino-propane - DTT dithiothreitol - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Mammalian cell genetics. 3. Characterization of x-ray-induced forward mutations in Chinese hamster cell cultures

X-irradiation induces forward mutations from 8-azaguanine sensitvity to resistance in Chinese hamster cells in culture. At this locus the number of induced mutations increases non-linearly with X-ray exposure. The mutation rate increase from 4.2·10⁻⁷ per locus per R with 200 R to 1.8·10⁻⁶ per locus per R with 1200 R. Several factors including cell density markedly influence the mutational yield. Reversion tests using specific chemical mutagens on 72 randomly isolated, azaguanine-resistant mutants suggest that both point mutations and chromosome deletions might have occurred in the hamster cells after exposure to ionizing radiation.     

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.     

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.     

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.     

Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK₁ cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

Alkamides from Echinacea inhibit cyclooxygenase-2 activity in human neuroglioma cells

During past years inhibition of the cyclooxygenase-2 (COX-2) enzyme has been proven as an effective strategy to suppress pain and inflammation. Based on this and other mechanistic findings, interest has also renewed in the molecular pathways underlying the anti-inflammatory effects of herbal drugs. The present study addressed this issue and investigated the impact of several polyunsaturated alkamides isolated from a CO2 extract of the roots of Echinacea angustifolia DC. on both activity and expression of COX-2. A 48-h treatment of H4 human neuroglioma cells with the CO2 extract led to a significant suppression of prostaglandin (PG) E2 formation. Analysis of eight different alkamides revealed a contribution of undeca-2Z-ene-8,10-diynoic acid isobutylamide (A5), dodeca-2E-ene-8,10-diynoic acid isobutylamide (A7), and dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide (A8) to this response. Using an established short-term COX-2 activity assay, all three alkamides were shown to interfere with COX-2 activity. In contrast, none of the COX-2-suppressing nor any other tested alkamide was found to inhibit COX-2 mRNA and protein expression. Instead, increased COX-2 mRNA and protein levels were registered in the presence of the CO2 extract and most of the analyzed alkamides which caused, however, no stimulation of PG formation. Overall, our results suggest that certain alkamides derived from E. angustifolia roots may contribute to the pharmacological action of the herbal extract by inhibiting COX-2-dependent PGE2 formation at sites of inflammation.