Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3 A resolution (Rwork of 17.8% and Rfree of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (alpha2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle.     

Comparison of urine cytology between the ileal conduit and Indiana pouch

OBJECTIVE: To compare the cytomorphologic features of urine obtained from two different kinds of urinary diversions constructed after total bladder resection. STUDY DESIGN: The smears of urine from 11 ileal conduits and 6 Indiana pouches were evaluated. All patients underwent total bladder resection due to transitional cell carcinoma (TCC) or other kinds of cancer before urine diversion. RESULTS: The cytologic features of Indiana pouch urine include degenerated, small, round cells without columnar cells derived from intestinal epithelium. In ileal conduit urine, well-preserved columnar cells and degenerated, small, round cells were frequently observed. The columnar cells in ileal conduit urine exhibited cytologic features that should be distinguished from TCC cells. CONCLUSION: The method of reconstructing the urinary tract is important in urine cytology from urine diversions because the cytomorphologic features of urine are different between the two kinds of urinary diversions. Since columnar cells in ileal conduit urine might lead to misdiagnosis as TCC, special consideration is required to examine ileal conduit urine.     

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21.

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.     

Regulation of cyclic GMP phosphodiesterase in the submandibular gland of adult rat

Cyclic GMP phosphodiesterase was investigated in the submandibular gland of the adult rat to elucidate the regulatory mechanisms of cGMP concentration in this gland. Ca2+ sensitivity was easily demonstrated as in other tissues using EGTA in the buffer for elution from DEAE-cellulose. The presence of inhibitor proteins for basal and calmodulin-activated portions of Ca2+-dependent phosphodiesterase was suggested. The inhibitor for the basal activity of Ca2+-dependent phosphodiesterase was deemed to be a heat-labile protein, which decreased Vmax, but had no effect on the Km value for cGMP. The presence of more than one kind of inhibitor for the calmodulin-activated portion of the Ca2+-dependent phosphodiesterase was also suggested. One of these, which was not absorbed on DEAE-cellulose, was a heat-labile protein which caused an increase of Km for cGMP, but no change of Vmax.     

Relationship between endothelin and thromboxane A2 in rat liver microcirculation.

In our previous study, we determined changes in hepatic blood flow using a Laser Doppler blood flow meter after i.v. injection of endothelin-1 (ET-1) or endothelin-3 (ET-3) at 2 nmol/kg in rats and found that ET-3 caused greater decreases in blood flow than ET-1. In the present study, we determined how the arachidonic acid cascade, mainly thromboxane A2 (TXA2), is related to ET-1 and ET-3 using indomethacin (INDO), which inhibits the biosynthesis of prostaglandin (PG), and OKY-046, a selective inhibitor of TXA2 synthesis. In the first series of experiments, ET-1 and ET-3 were administered after inhibiting the biosynthesis of PG by s.c. injection of 2 mg/kg of INDO. While INDO failed to inhibit the slight decrease in hepatic blood flow induced by ET-1, it significantly inhibited the marked decrease in hepatic blood flow elicited by ET-3. In the next series of experiments, ET-1 and ET-3 were administered after administration of 20 mg/kg of OKY-046. OKY-046 showed no effects in animals treated with ET-1, as in those pre-treated with INDO, while it significantly inhibited the decreases in hepatic blood flow induced by ET-3. These findings suggest that ET-1 decreases hepatic blood flow due to its direct effects although to a lesser extent than ET-3, while ET-3 does so due not only to its direct effects but also to TXA2-mediated effects. It is therefore likely that in addition to ET family peptides, PG-mediated mechanisms are involved in the regulation of hepatic microcirculation by ETs.     

Stimulation of pituitary-adrenocortical system by ginseng saponin.

Effects of preparations of saponin mixture and isolated ginsenosides, extracted from the root of Panax ginseng, on plasma corticotropin (ACTH) and corticosterone concentrations in rats were determined by the radioimmunoassay and competitive protein binding method. When ginseng saponin mixture was administered to rats intraperitoneally, plasma ACTH and corticosterone increased significantly 30, 60 and 90 min after the treatment. The kinetic pattern of the increase in plasma ACTH was almost parallel to that in plasma corticosterone. Isolated ginsenoside, protopanaxadiol or protopanaxatriol glycoside, also increased plasma corticosterone. The ginseng-induced increase in plasma corticosterone was suppressed by pretreatment with dexamethasone. Thus the ginseng saponin was found to act on the hypothalamus and/or hypophysis primarily, and stimulated ACTH secretion which resulted in increased synthesis of corticosterone in the adrenal cortex.     

Binding of bacterial lipopolysaccharide to histocompatibility-2-complex proteins of mouse lymphocytes.

The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Cell expansion and microfibril deposition inClosterium ehrenbergii

Cells ofClosterium ehrenbergii expanded for about 4.5 hr after vegetative cell division and about 3 hr after sexual cell division. Short cells were produced by the sexual expansion. At the early stage of the vegetative and the sexual expansion, most microfibrils were deposited transversely to the cell axis on the inner surface of the new wall. Then, bundles of microfibrils, running in various directions, overlaid the transverse microfibrils already deposited, at the late stage of the both types of expansion. The pattern of microfibril deposition changed from the transverse to the bundled pattern about 4 hr after the vegetative and about 2 hr after the sexual cell division, respectively. The change of pattern of microfibril deposition seemed to be highly correlated with cessation of cell expansion.