Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line

The Na⁺ pump (Na⁺, K⁺-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na⁺ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 ± 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, ¹⁴C-taurine efflux increased by 183 ± 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a ¹⁴C-taurine efflux of 207 ± 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic α isoforms of Na⁺, K⁺-ATPase demonstrated high levels of the ubiquitously expressed α1 and the neuronal-specific α3. Ouabain-binding data showed that CHP-100 cells express ∼3 × 10⁵ pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na⁺, K⁺-ATPase in CHP-100 cells. J. Cell. Physiol. 174:145–153, 1998. © 1998 Wiley-Liss, Inc.     

Guanine deaminase inhibitor from rat liver. Isolation and characterization

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.     

Exogenous and endogenous increase in fungal GGPP increased fungal Taxol production

Applied Microbiology and Biotechnology - Taxol is an anticancer identified in both endophytic fungus and its host plant. Plant Taxol is a diterpenoid with geranylgeranyl diphosphate (GGPP) mediates...     

Applying multi-criteria decision-making on alternatives for earth-retaining walls: LCA,LCC, and S-LCA

The International Journal of Life Cycle Assessment - The aim of this research is to assess the sustainability performance of different concrete and stone walls being used in residential buildings...     

Locomotion of an efficient biomechanical sperm through viscoelastic medium

Biomechanics and Modeling in Mechanobiology - Every group of microorganism utilizes a diverse mechanical strategy to propel through complex environments. These swimming problems deal with the...     

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V_max = 2.31 pmol min^?1 U^?1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.