Homocysteine transport by human aortic endothelial cells: identification and properties of import systems

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Transport of L-homocysteine into and out of the human vascular endothelium is poorly understood. We hypothesized that cultured human aortic endothelial cells (HAEC) would import L-homocysteine on one or more of the L-cysteine transport systems. Inhibitors of the transporters were used to characterize the uptake of [35S]L-homocysteine, [35S]L-homocystine, and [35S]L-cysteine. We found that L-homocysteine uptake is mediated by the sodium-dependent cysteine transport systems X(AG), ASC, and A, and the sodium-independent transport system L. Thus, HAEC utilize multiple cysteine transporters (X(AG) > or = L > ASC > A) to import L-homocysteine. Kinetic analysis supported the uptake results. Michaelis-Menten constants (Km) for the four systems yielded values of 19.0, 27.1, 112, and 1000 microM for systems L, X(AG), ASC, and A, respectively. The binding and uptake of [35S]L-homocystine, the disulfide homodimer of L-homocysteine, was mediated by systems X(AG), L, and ASC but not by system A. In contrast to [35S]L-homocysteine, system x(c) was active for [35S]L-homocystine uptake. A similar pattern was observed for [35S]L-cysteine. Thus, L-homocysteine and L-homocystine found in hyperhomocysteinemic subjects can gain entry into the vascular endothelium by way of multiple L-cysteine transporters.     

WISP-2/CCN5 is involved as a novel signaling intermediate in phorbol ester-protein kinase Calpha-mediated breast tumor cell proliferation

PMA and active phorbol esters stimulate the proliferation of various tumor cells, including ER-positive human breast tumor cell lines. However, the specific signaling pathways involved in the PMA-induced mitogenic effect on breast tumor cells have not been fully elucidated. In the present study, we explored the mechanisms associated with the mitogenic influence of PMA on breast tumor cells. Following an acute exposure (i.e., within 2 to 6 h) to PMA (50 nM), a mitogenic effect was observed on WISP-2/CCN5-positive breast tumor cell lines, including MCF-7, ZR-75-1 and SKBR-3 cells, and induction of WISP-2/CCN5 mRNA expression paralleled the observed mitogenic proliferation. This effect was undetected in WISP-2/CCN5 negative MDA-MB-231 breast tumor cells or human mammary epithelial cells with or without ER-alpha transfection. The mitogenic effect of PMA was perturbed by short hairpin RNA (shRNA)-mediated inhibition of WISP-2/CCN5 signaling in MCF-7 cells. Moreover, the upregulation of WISP-2/CCN5 by PMA is not ER dependent but is instead mediated through a complex PKCalpha-MAPK/ERK and SAPK/JNK signaling pathway, which leads to growth stimulation of MCF-7 breast tumor cells. These series of experiments provide the first evidence that WISP-2/CCN5 is a novel signaling molecule that critically participates in the mitogenic action of PMA on noninvasive, WISP-2/CCN5-positive breast tumor cells through PKCalpha-dependent, multiple molecular signal transduction pathways.     

Modulation of cell-cycle regulatory signaling network by 2-methoxyestradiol in prostate cancer cells is mediated through multiple signal transduction pathways

2-Methoxyestradiol (2-ME(2)), a promising anticancer drug, induces growth arrest and apoptosis in various androgen-dependent (LNCaP) and -independent (DU145 and PC-3) prostate cancer cell lines. Moreover, flow cytometric analysis indicated a novel dual impact of 2-ME(2) on the cell division cycle of prostate cancer cells. Chronic exposure of high doses of 2-ME(2) enhance the accumulation of cells in S and G2/M phases, while cell numbers in the G1 phase were reduced significantly by this treatment. Because cyclin B1 overexpression, induction of cdc2 phosphorylation, and its regulatory proteins wee1 and phospho-cdc25C (interphase and mitotic forms) by 2-ME(2) treatment correlated with the induction of apoptosis, growth arrest at the G2/M phase, and accumulation of the S phase, we reasoned that cyclin B1 and cdc2 phosphorylation and its upstream regulatory molecular networks may be associated with the ultimate impacts of 2-ME(2). Because phosphorylation of cdc2 and upregulation of wee1 by 2-ME(2) can be abolished by both extracellular receptor kinase (ERK) inhibitor (U0126) and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), our studies indicate that the 2-ME(2)-induced upregulation of wee1 and subsequent cdc2 phosphorylation are mediated through mitogen-activated protein kinase (MAPK)-ERK-JNK signaling pathways.     

GTP-induced conformational changes in translin: a comparison between human and Drosophila proteins

Human translin is a conserved protein, unique in its ability to bind both RNA and DNA. Interestingly, GTP binding has been implicated as a regulator of RNA/DNA binding function of mouse translin (TB-RBP). We cloned and overexpressed the translin orthologue from Drosophila melanogaster and compared its DNA/RNA binding properties in relation to GTP effects with that of human protein. Human translin exhibits a stable octameric state and binds ssDNA/RNA/dsDNA targets, all of which get attenuated when GTP is added. Conversely, Drosophila translin exhibits a stable dimeric state that assembles into a suboctameric (tetramer/hexamer) form and fails to bind ssDNA and RNA targets. Interestingly enough, CD spectral analyses, partial protease digestion profile revealed GTP-specific conformational changes in human translin, whereas the same were largely missing in Drosophila protein. Isothermal calorimetry delineated specific heat changes associated with GTP binding in human translin, which invoked subunit "loosening" in its octamers; the same effect was absent in Drosophila protein. We propose that GTP acts as a specific molecular "switch" that modulates the nucleic acid binding function selectively in human translin, perhaps by affecting its octameric configuration.     

Asymmetric epoxidation of alkenes using a mixed-ligand complex of ruthenium(III) containing a sugar-based ligand

A mixed-ligand ruthenium(III) catalyst complex, [Ru^III(TDL*)(bipy)(H₂O)]Cl (1) (TDL* = N-3,5-di-(t-butyl)salicylidine-d-glucosamine; bipy = 2,2′-bipyridine) exhibited catalytic activity toward enantioselective alkene epoxidation using tert-butyl hydroperoxide as terminal oxidant. Styrene, 4-chlorostyrene, 4-methylstyrene, 4-methoxystyrene, 1-methylcyclohexene and 1,2-dihydronaphthalene were effectively converted to their organic epoxides with moderate enantioselectivity (37-47% ee) at ambient temperature. A mechanism involving the formation of a high-valent Ru(V)-oxo species, and the subsequent oxo-transfer to the alkene through a metallaoxetane intermediate is proposed.     

Topoisomerases of kinetoplastid parasites: why so fascinating?

DNA topoisomerases are the key enzymes involved in carrying out high precision DNA transactions inside the cells. However, they are detrimental to the cell when a wide variety of topoisomerase-targeted drugs generate cytotoxic lesions by trapping the enzymes in covalent complexes on the DNA. The discovery of unusual heterodimeric topoisomerase I in kinetoplastid family added a new twist in topoisomerase research related to evolution, functional conservation and their preferential sensitivity to Camptothecin. On the other hand, structural and mechanistic studies on kinetoplastid topoisomerase II delineate some distinguishing features that differentiate the parasitic enzyme from its prokaryotic and eukaryotic counterparts. This review summarizes the recent advances in research in kinetoplastid topoisomerases, their evolutionary significance and the death of the unicellular parasite Leishmania donovani induced by topoisomerase I inhibitor camptothecin.     

EHD4 and CDH23 Are Interacting Partners in Cochlear Hair Cells

Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.Hair cells located in the mammalian inner ear transform mechanical stimuli into electrical signals that in turn facilitate neurotransmitter release onto auditory neurons. The key element in the transduction process is the mechano-electric transducer (MET)² apparatus located near the top of the stereocilium. CDH23 is a single pass transmembrane protein with 27 extracellular cadherin repeats. It is one of the components of the tip-link (1, 2), which connects the top of a shorter stereocilium to the side of its taller neighbor (3). Vibrations of the basilar membrane of the inner ear ultimately result in deflection of the hair bundles, which modulates tension on the tip-link, thereby controlling the opening probability of cation-selective MET channels (3, 4). Cations, principally K⁺ and Ca²⁺, flow through the MET channels and ultimately change the membrane potential. A mutation in the gene encoding CDH23, the Usher syndrome type 1D factor (USH1D), causes deaf-blindness in humans (5). Several interacting partners of CDH23 have been reported and include another tip-link protein protocadherin 15 (6), a multi-PDZ domain-containing scaffold protein harmonin (7) and a stereociliary scaffolding protein MAGI-1 (8). Protocadherin 15 binds to CDH23 through its extracellular domains (6), whereas the cytoplasmic region of CDH23 interacts with MAGI-1 and harmonin through its PDZ domain-binding interfaces (PBI). Harmonin also associates with other USH1 factors like myosin VIIa, protocadherin 15, and sans (9). These findings indicate that harmonin bridges CDH23 to the cytoskeletal actin core of the stereocilium and is probably essential for the developmental differentiation of stereocilia (10–12). However, it is currently unknown how CDH23 is transported to the tip of stereocilia. To search for additional interacting partners of CDH23, we performed a membrane-based yeast two-hybrid assay, which identified EHD4 as a potential binding partner (13).EHD4 belongs to an evolutionarily conserved EH (Eps 15 homology) domain-containing protein family involved in endocytic trafficking and recycling. Four highly homologous members of this family, EHD1–4, are expressed in mammalian cells. They contain a single C-terminal EH domain, an N-terminal nucleotide-binding loop and a coiled-coil region responsible for oligomerization (14–16). Of the four EHD proteins EHD1 is the best characterized and is involved in regulating the recycling of membrane receptors including the transferrin receptor and the major histocompatibility complex class I (17, 18). EHD1 is also involved in controlling cholesterol recycling and homeostasis (19) and in facilitating endosome to Golgi retrieval (20). EHD3 appears to regulate receptor movements from the early endosome (EE) to the endocytic recycling compartment (ERC) and Golgi (21, 22). EHD2 was isolated from GLUT4-enriched fractions of adipocytes and was shown to regulate insulin-mediated translocation of GLUT4 to the plasma membrane (23, 24). Additionally, EHD2 is involved in the regulation of transferrin receptor internalization (23), recycling (25), and actin cytoskeleton rearrangement (23). EHD4, also called Pincher, was first reported as an extracellular matrix protein (26). Subsequent studies have shown this intracellular protein to be involved in the regulation of neurotrophin receptor TrkA endocytosis in pheochromocytoma (PC12) cells (27). It is also involved in interactions with the cell fate determinant, NUMB, and co-localizes with the small GTP-binding protein, Arf6 (28). Recently, Sharma et al. (29) showed that EHD4 regulates the exit of endocytic cargo from the early endosome toward both the recycling compartment and the late endocytic pathway. They also indicated that EHD4 and EHD1 interact transiently as most of the EHD4 resides on peripheral early endosomes, while EHD1 resides primarily on tubular recycling compartments. This partial overlap/association might be necessary for the transport of proteins through the early endosome to the ERC. Previously, George et al. (25) had also demonstrated that EHD4 interacts with EHD1 and its paralogs, which suggests cooperation and partial overlap of function between EHD4 and EHD1.Unlike other CDH23-binding proteins, EHD4 does not contain a PDZ domain that could bind to the PBI located in the cytoplasmic tail of CDH23. In addition, the cytoplasmic tail of CDH23 lacks an Asn-Pro-Phe (NPF) motif that could mediate an interaction with the EH domain of EHD4. Therefore, we proceeded to characterize the authenticity of interaction between EHD4 and CDH23 identified in yeast and mammalian cells, using both in vitro and in vivo methods. We verified the expression of EHD4 mRNA in mouse cochlea and investigated the physiological role of EHD4 protein in the cochlea using EHD4-KO mice.     

Alterations in N-methyl-D-aspartate receptor subunits in primary sensory neurons following acid-induced esophagitis in cats

The excitatory amino acid glutamate plays an important role in the development of neuronal sensitization and the ionotropic N-methyl-d-aspartate receptor (NMDAR) is one of the major receptors involved. The objective of this study was to use a cat model of gastroesophageal reflux disease (GERD) to investigate the expression of the NR1 and NR2A subunits of NMDAR in the vagal and spinal afferent fibers innervating the esophagus. Two groups of cats (Acid-7D and PBS-7D) received 0.1 N HCl (pH 1.2) or 0.1 M PBS (pH 7.4) infusion in the esophagus (1 ml/min for 30 min/day for 7 days), respectively. NR1 splice variants (both NH(2) and COOH terminals) and NR2A in the thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophagus were evaluated by RT-PCR, Western blot, and immunohistochemistry. Acid produced marked inflammation and a significant increase in eosinophil peroxidase and myeloperoxidase contents compared with PBS-infused esophagus. The NR1-4 splice variant gene exhibited a significant upregulation in DRGs and esophagus after acid infusion. In DRGs, NGs, and esophagus, acid infusion resulted in significant upregulation of NR1 and downregulation of NR2A subunit gene expression. A significant increase in NR1 polypeptide expression was observed in DRGs and NGs from Acid-7D compared with control. In conclusion, long-term acid infusion in the cat esophagus resulted in ulcerative esophagitis and differential expressions of NR1 and NR2A subunits. It is possible that these changes may in part contribute to esophageal hypersensitivity observed in reflux esophagitis.     

Nitric oxide and dihydrolipoic acid modulate the activity of caspase 3 in HepG2 cells

Herein, we report that dihydrolipoic acid and lipoic acid (LA) plus lipoamide dehydrogenase and NADH denitrosate S-nitrosocaspase 3 (CASP-SNO). In HepG2 cells, S-nitroso-l-cysteine ethyl ester (SNCEE) impeded the activity of caspase 3 (CASP-SH), while a subsequent incubation of the cells in SNCEE-free medium resulted in endogenous denitrosation and reactivation of CASP-SH. The latter process was inhibited in thioredoxin reductase-deficient HepG2 cells, in which, however, LA markedly reactivated CASP-SH. The data obtained are discussed with focus on low molecular mass dithiols that mimic the activity of thioredoxin in reactions of protein S-denitrosation.