Kupffer cell-mediated down regulation of rat hepatic CMOAT/MRP2 gene expression

Lipopolysaccharides (LPS) induces intrahepatic cholestasis and canalicular multispecific organic anion transporter (CMOAT/MRP2) plays a central role in hepatic bilirubin transport. This study examined the role of Kupffer cell in LPS-induced cholestasis. Rats were injected intravenously with LPS. Kupffer cells were inactivated with gadolinium chloride (Gd). CMOAT/MRP2 mRNA expression was time- and dose-dependently decreased by LPS injection with a decrease in bile flow and an increase in serum bilirubin level. Gd pretreatment inhibited decrease in CMOAT/MRP2 mRNA and bile flow, and increase in serum bilirubin. Kupffer cell-conditioned medium decreased CMOAT/MRP2 expression. Addition of anti-IL-1 or anti-TNFalpha antibody restored CMOAT/MRP2 expression, whereas IL-1 and TNFalpha decreased the expression. MAP kinases were activated by addition of the conditioned medium, and addition of PD98059 or SB203580 restored CMOAT/MRP2 expression. These results suggest that LPS activates Kupffer cells to secrete IL-1 and TNFalpha, which in turn activate MAP kinases and decrease CMOAT/MRP2 expression.     

An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions

We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer. A genomewide search showed that E. histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E. histolytica. The EhNifS protein expressed in E. coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms. Eh-NifU protein existed as a tetramer and contained one stable [2Fe-2S]2+ cluster per monomer, revealed by spectroscopic and iron analyses. Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase. Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes.     

8-Hydroxydaidzein, an aldose reductase inhibitor from okara fermented with Aspergillus sp. HK-388

The aldose reductase (AR) inhibitor, 8-hydroxydaidzein, was isolated and identified from a methanolic extract of okara (soybean pulp) fermented with the fungal strain, Aspergillus sp. HK-388. 8-Hydroxydaidzein showed non-competitive inhibition of human recombinant AR with respect to DL-glyceraldehyde, its Ki value being evaluated as 7.0 microM.     

Theoretical evidence that more microtubules reach the cortex at the pole than at the equator during anaphase in sea urchin eggs

Astral microtubules are rapidly elongated during anaphase and telophase in sea urchin eggs. The number of microtubules extending to the cell surface was calculated with a computer. For the calculations, microtubules were assumed to radiate from the astral center uniformly over angles. Although microtubules from two asters freely overlapped around the equator, the number per the unit area, i.e. the surface density, was larger in the polar region than in the equatorial region. The ratio of the theoretically calculated numbers in the two regions was close to the ratio obtained from the ultrastructural observations by Asnes and Schroeder in 1979. When counted in the longitudinal section including two astral centers, the microtubule number was a little larger in the equatorial region than in the polar region. However, the numbers do not represent the surface density because the two-dimensional section contains only a small portion of all the microtubules spreading in the three-dimensional space. The fluorescence image for tubulin, in most cases, provides the microtubule distribution in the longitudinal section. Therefore, from such an image, we cannot judge whether the surface density of astral microtubules is larger at the pole or at the equator.     

Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase

Isoflavonoids are distributed predominantly in leguminous plants, and play pivotal roles in the interaction of host plants with biological environments. Isoflavones in the diet also have beneficial effects on human health as phytoestrogens. The isoflavonoid skeleton is constructed by the CYP93C subfamily of cytochrome P450s in plant cells. The reaction consists of hydroxylation of the flavanone molecule at C-2 and an intramolecular 1,2-aryl migration from C-2 to C-3 to yield 2-hydroxyisoflavanone. In this study, with the aid of alignment of amino acid sequences of CYP93 family P450s and a computer-generated putative stereo structure of the protein, candidates for key amino acid residues in CYP93C2 responsible for the unique aryl migration in 2-hydroxyisoflavanone synthase reaction were identified. Microsomes of recombinant yeast cells expressing mutant proteins of CYP93C2 were prepared, and their catalytic activities tested. The reaction with the mutant in which Ser 310 in the centre of the I-helix was converted to Thr yielded increased formation of 3-hydroxyflavanone, a by-product of the 2-hydroxyisoflavanone synthase reaction, in addition to the major isoflavonoid product. More dramatically, the mutant in which Lys 375 in the end of beta-sheet 1-4 was replaced with Thr produced only 3-hydroxyflavanone and did not yield the isoflavonoid any longer. The roles of these amino acid residues in the catalysis and evolution of isoflavonoid biosynthesis are discussed.     

Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Expression of mRNAs for Neurotrophins (NGF,BDNF, and NT-3) and their Receptors (p75NGFR,Trk, TrkB,and TrkC) in Human Peripheral Neuropathies

The steady-state mRNA levels of NGF, BDNF and NT-3, and the mRNA levels of their receptors p75^NGFR, trk, trk,B and trkC were examined in various human peripheral neuropathies, to determine the correlation with myelinated fiber pathology and T cell and macrophage invasions in the diseased nerves. Steady state levels of p75^NGFR mRNAs were significantly elevated in nerves with axonal pathology. In contrast, steady state levels of trkB and trkC mRNA levels were diminished, trk mRNA was not detected in the human nerves. The NGF, BDNF, and NT-3 mRNA levels were elevated in the diseased nerves. The increase in BDNF and NT-3 mRNA levels were proportional to the extent of invasion of the nerves by T cells and macrophages, but did not directly correlate with axonal nor demyelinating pathology, thus suggesting that inflammatory cell invasions are involved in the regulation of BDNF and NT-3 mRNA expressions. These neurotrophin and their receptor gene expressions in the diseased human nerves would be regulated by an underlying pathology-related process, and could play a role in peripheral nerve repair.