Molecular cloning,expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis

PCR techniques were used to clone and identify cDNAs for ecdysone receptor A and B1 (EcR-A and EcR-B1) isoforms from the rice stem borer, Chilo suppressalis. They differ only in the N-terminal A/B regions and show high sequence identities to other insects' EcRs. At the wandering stage, EcR-B1 mRNA was expressed more abundantly in the midgut than in the epidermis and fat body, whereas expression levels of EcR-A mRNA were similar in the three tissues. In the epidermis of the last instar larvae, the maximal mRNA expression of both EcR-A and EcR-B1 was observed from the wandering to prepupal stages prior to the peak of ecdysteroid titer in the hemolymph. In gel mobility shift assays, in vitro translated C. suppressalis EcR-B1 (CsEcR-B1) and Bombyx mori ultraspiracle (BmUSP) proteins bound to the Pal 1 and Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. These results indicate that the cDNAs isolated here encode functional ecdysone receptors.     

Inositol phospholipids cause the activation of adrenal tyrosine hydroxylase through their electrostatic action on the enzyme

The effects of inositol phospholipids on adrenal tyrosine hydroxylase (TH) were studied. Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) caused a rapid and concentration-dependent activation of TH in vitro. The potency of this activation was dependent on the number of phosphate groups in the lipid molecule, and the activation was completely suppressed by increasing the concentrations of salts in the reaction mixture. These results seem to indicate that the activation of TH by inositol phospholipids may be due to their electrostatic action, and suggest a possible involvement of inositol phospholipids in the regulation of TH activity in vivo.     

Restoring catalytic activity to the human carbonic anhydrase (CA) related proteins VIII,X and XI affords isoforms with high catalytic efficiency and susceptibility to anion inhibition

Mutation of amino acid residues 94, 96 and 119 to histidine(s) in the human carbonic anhydrase (CA, EC 4.2.1.1) related proteins CARP VIII, X and XI restored the zinc binding and catalytic activity for the hydration of CO₂ to bicarbonate. CA VIII, X and XI thus obtained showed high catalytic activity (67.3–92.0% of the activity of hCA II and much higher compared to hCA I) and were inhibited in the milli-micromolar range by inorganic anions, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid. Among the three new isoforms, hCA X was the most efficient enzyme and also showed the highest affinity for anion inhibitors (K_Is of 3.6–68 μM for phenylboronic acid, sufamic acid, sulfamide, cyanide and azide). hCA VIII was poorly inhibited by halides, cyanate, nitrate and sulfate (K_Is of 38.4–65.4 mM), whereas CA XI had a behavior intermediate between that of hCA VIII and X, both regarding the catalytic activity and sensitivity to anion inhibitors.     

Iron reduction by deferoxamine leads to amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes KKAy mice

Iron is an essential trace metal for most organisms. However, excess iron causes oxidative stress through production of highly toxic hydroxyl radicals via the Fenton/Haber-Weiss reaction. Iron storage in the body is reported to be associated with fat accumulation and type 2 diabetes mellitus. We investigated the role of iron in adiposity by using KKAy mice and obese and diabetic model mice. Eight-week-old KKAy mice were divided into two groups and treated with deferoxamine (DFO), an iron chelator agent, or a vehicle for 2 wk. DFO treatment diminished fat iron concentration and serum ferritin levels in KKAy mice. Fat weight and adipocyte size were reduced significantly in DFO-treated mice compared with vehicle-treated mice. Macrophage infiltration into fat was also decreased in DFO-treated mice compared with vehicle-treated mice. Superoxide production and NADPH oxidase activity in fat, as well as urinary 8-hydroxy-2'-deoxyguanosine excretion, were decreased in KKAy mice after DFO treatment while p22(phox) expression in adipose tissue was diminished in such mice. Ferritin expression in the fat of DFO-treated KKAy mice was decreased. In addition, F4/80-positive cells also presented through both p22(phox) and ferritin expression. The mRNA expression levels of inflammatory cytokines were also reduced in fat tissue of DFO-treated mice. These findings suggest that reduction of iron levels ameliorates adipocyte hypertrophy via suppression of oxidative stress, inflammatory cytokines, and macrophage infiltration, thereby breaking a vicious cycle in obesity.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

Identification and characterization of SEB, a novel protein that binds to the acute undifferentiated leukemia-associated protein SET.

SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is a 39-kDa nuclear phosphoprotein and has an inhibitory activity for protein phosphatase 2A (PP2A). SET is fused to a putative oncoprotein, CAN/NUP214, in AUL and is thought to play a key role in leukemogenesis by its nuclear localization, protein-protein interactions and PP2A inhibitory activity. Here, we describe the isolation and characterization of a novel cDNA encoding a protein with 1542 amino-acid residues that specifically interacts in a yeast two-hybrid system as well as in human cells with SET. This new protein, which we name SEB (SET-binding protein), is identified as a 170-kDa protein by immunoprecipitation with a specific antibody and is localized predominantly in the nucleus. SEB1238--1434 is determined as a SET-binding region that specifically binds to SET182--223. SEB also has an oncoprotein Ski homologous region (amino acids 654--858), six PEST sequences and three sequential PPLPPPPP repeats at the C-terminus. SEB mRNA is expressed ubiquitously in all human adult tissues and cells examined. The SEB gene locus is assigned to the chromosome 18q21.1 that contains candidate tumor suppressor genes associated with deletions in cancer and leukemia. Although the function of SEB is not known, we propose that SEB plays a key role in the mechanism of SET-related leukemogenesis and tumorigenesis, perhaps by suppressing SET function or by regulating the transforming activity of Ski in the nucleus.     

GRAY WHALES (ESCHRICHTIUS ROBUSTUS) OFF SAKHALIN ISLAND,RUSSIA: SEASONAL AND ANNUAL PATTERNS OF OCCURRENCE1

The annual return, seasonal occurrence, and site fidelity of Korean-Okhotsk or western gray whales on their feeding grounds off northeastern Sakhalin Island, Russia, were assessed by boat-based photo-identification studies in 1994-1998. A total of 262 pods were observed, ranging in size from 1 to 9 whales with an overall mean of 2.0. Sixty-nine whales were individually identified, and a majority of all whales (71.0%) were observed in multiple years. Annual sighting frequencies ranged from 1 to 18 d, with a mean of 5. 4 d. The percentage of whales reidentified from previous years showed a continuous annual increase, reaching 87.0% by the end of the study. Time between first and last sighting of identified individuals within a given year was 1-85 d, with an overall mean of 40.6 d. Annual calf proportions ranged from 4.3% (1997) to 13.2% (1998), and mother-calf separations generally occurred between July and September. The seasonal site fidelity and annual return of whales to this part of the Okhotsk Sea emphasize its importance as a primary feeding ground for this endangered population.