Cloning and expression of a cDNA encoding mouse indoleamine 2,3-dioxygenase

The depletion of an essential amino acid (aa), tryptophan, caused by interferon-gamma (IFN-gamma)-mediated induction of indoleamine 2,3-dioxygenase (IDO) in mouse allografted tumor cells, has been suggested as a reason for the allograft rejection. To elucidate the mechanism of this IDO induction, attempts were made to isolate cDNA clones encoding mouse IDO. In seven of 25 mouse cell lines, IDO was induced by IFN-gamma, and the highest IDO induction was observed in the case of rectal cancer (CMT-93) cells, which were further stimulated two- to threefold by the simultaneous addition of dibutyryl cyclic AMP (Bt2cAMP). A cDNA library was prepared from poly(A)+ RNA isolated from CMT-93 cells treated with IFN-gamma/Bt2cAMP. The cDNA clones were isolated using the cDNA encoding human IDO as a probe. The mouse IDO cDNA encodes a 407-aa protein with an Mr of 45,639. The deduced aa sequence agreed with partial aa sequences derived from endopeptidase digestion of purified mouse IDO and revealed 61% homology with that of human IDO. Transient expression of the mouse IDO cDNA in COS-7 cells yielded a high level of IDO activity in the cells. Northern hybridization analysis of RNA in CMT-93 cells indicated that IFN-gamma induced the IDO mRNA, and that the level of RNA was increased by simultaneous addition of Bt2cAMP, while Bt2cAMP itself had no effect on mRNA induction.     

Comparison of the affinities of newly identified human bile acid binder and cationic glutathione S-transferase for bile acids

The bile acid binding properties of the newly identified bile acid binder (Mr = 36,000) (FEBS Lett. 1984. 177: 31-35) and the major cationic glutathione (GSH) S-transferase (Mr = 50,000) in human liver cytosol were compared. Binding affinities were measured by the competitive displacement by bile acids of 1-anilino-8-naphthalene sulfonate (ANS) bound to the proteins and, in some cases, by direct methods of flow dialysis and equilibrium dialysis. The binding affinities for various bile acids by the human bile acid binder were 2-5 orders of magnitude greater than those by human cationic GSH S-transferase. This suggests an important physiologic role for the former protein in intracellular transfer of bile acids in human liver.     

Human indolylamine 2,3-dioxygenase. Its tissue distribution, and characterization of the placental enzyme.

The presence of indolylamine 2,3-dioxygenase was examined in human subjects by determining its activity with L-tryptophan as substrate. Enzyme activity was detected in various tissues, and was relatively high in the lung, small intestine and placenta. Human indolylamine 2,3-dioxygenase, partially purified from the placenta, had an Mr of about 40 000 by gel filtration and exhibited a single pI of 6.9. The human enzyme required a reducing system, ascorbic acid and Methylene Blue, for maximal activity and was able to oxidize D-tryptophan, 5-hydroxy-L-tryptophan as well as L-tryptophan, but kinetic studies indicated that the best substrate of the enzyme was L-tryptophan.     

IFN-gamma is the inducer of indoleamine 2,3-dioxygenase in allografted tumor cells undergoing rejection

The depletion of an essential amino acid, tryptophan, caused by induction of indoleamine 2,3-dioxygenase (IDO), has been shown to be a mechanism involving self-defense against inhaled microorganisms and tumor growth. We recently reported that the IDO is dramatically (approximately 50-fold) induced in allografted tumor (3-methylcholanthrene-induced ascites type tumor cells) cells undergoing rejection, and that the enzyme is induced by factor(s) released through the interaction of allografted tumor cells with infiltrating leukocytes. The culture supernatant of infiltrating leukocytes, which were harvested on day 7 after tumor transplantation, induced the highest IDO activity in the tumor cells. The inducer activity was completely neutralized by the addition of antibody to IFN-gamma but not by antibody to IFN-alpha/beta. Approximately 6 U/ml of IFN-gamma was detected by an ELISA assay in the 12-h culture supernatant with 2 x 10(6) leukocytes/ml, and rIFN-gamma at 6 U/ml induced IDO in 3-methylcholanthrene-induced ascites type tumor cells to the same extent as IFN-gamma in the culture supernatant. Moreover, i.p. administration of antibody to IFN-gamma almost completely inhibited the induction of IDO in the allografted tumor cells. These observations indicate that the factor responsible for IDO induction in the allografted tumor cells is IFN-gamma.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

Semilunar postlarval settlement periodicity ensuing from semilunar larval release cycle in the Nihonotrypaea harmandi (Crustacea,Decapoda, Axiidea,Callianassidae) population on an intertidal sandflat in an open marine coastal embayment

Many decapod crustaceans in marine intertidal habitats release larvae toward coastal oceans, from which postlarvae (decapodids: settling-stage larvae) return home. Decapodid settlement processes are poorly understood. Previous studies showed that in Kyushu, Japan, the callianassid shrimp population on an intertidal sandflat of an open bay joining the coastal ocean near a large estuary released eight batches of larvae basically in a semilunar cycle from June through October and that decapodids performed diel vertical migration, occurring in the water column nocturnally. We conducted (a) frequent sampling for population density and size-composition on the sandflat through one reproductive season, (b) planktonic and benthic sampling for decapodids around the bay mouth, and (c) current meter deployment at three points across the bay mouth for tidal harmonic analysis. On the sandflat, six batches of newly-settled decapodids (settlers) occurred in a semilunar periodicity until October, with peaks occurring 0–3 days before syzygy dates except for the first one. For larval Batches 1–4, buoyancy-driven shoreward subsurface currents during July to mid-October would transport some pre-decapodid-stage larvae (zoeae) toward the bay. The absence of expected settler Batches 7–8 would be due to the converse subsurface currents caused by water-column mixing and seasonal winds after mid-October, carrying zoeae offshore. Once in the bay, phasing of night and nighttime-averaged shoreward tidal current explained the settlement pattern for Batches 1–4. For Batches 5–6 occurring in mid-September to mid-October, water currents generated by seasonal wind and tidal forcings may have caused peak settlement after the time expected from tidally-driven decapodid transport.     

Effects of Zinc Acetate on Serum Zinc Concentrations in Chronic Liver Diseases: a Multicenter,Double-Blind,Randomized, Placebo-Controlled Trial and a Dose Adjustment Trial

Biological Trace Element Research - The essential trace element zinc maintains liver functions. Liver diseases can alter overall zinc concentrations, and hypozincemia is associated with various...     

Close Genetic Relationship between Legionella pneumophila Serogroup 1 Isolates from Sputum Specimens and Puddles on Roads,as Determined by Sequence-Based Typing

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires'' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.