Battery of tests for profiling abnormalities of vitamin K-dependent coagulation factors in drug-toxicity studies in rats.

A battery of simple tests for profiling abnormalities of vitamin K-dependent coagulation factors encountered in drug-toxicity studies was verified in rats treated with warfarin (3 and 10 mg/kg, p.o). The thrombotest, or hepaplastin-test, is useful as a follow-up test after routine screening tests for coagulation abnormalities based on PT and APTT, to rule out other coagulation-factor abnormalities. Measurement of coagulation factor activities (factors II, VII, IX and X) using factor-deficient human plasmas provides direct evidence of decreased activities of vitamin K-dependent factors. Furthermore, Echis carinatus venom coagulation time, together with factor II activity, allows us to confirm the generation of PIVKA-II.     

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES*

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH₂. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.     

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission

Iminodiacetate–DNA conjugates and acridine–DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)–iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine–DNA conjugate.Electronic Supplementary Material Supplementary material is available for this article at .     

Structure of the partially unliganded met state of 400 kDa hemoglobin: insights into ligand-induced structural changes of giant hemoglobins

Recent crystallographic studies have revealed the structures of some invertebrate extracellular giant hemoglobins of 3,600 kDa or 400 kDa and their common quaternary structure of dodecameric subassembly composed of four kinds of globin subunits (A1, A2, B1, and B2). These results have provided insight into the mechanisms of their unique functional properties of oxygen binding and sulfide binding. All of these structures were solved with oxygenated or CO-liganded forms at low or moderate resolutions. We have determined the crystal structure of 400 kDa Hb from a polychaete Oligobrachia mashikoi at 1.95 A resolution. The electron densities at higher resolution confirm the existence of an isoform of the B1 subunit because of the inconsistency with the model that was built from the formerly known amino acid sequence. The brownish color of the crystals used in this study and the absorption spectrum from the dissolved crystals strongly indicated that the obtained structure was a ferric met state, whereas complete absence of electron density around the distal heme pockets were observed at the A2, B1, and B2 subunits. We concluded that the obtained structure was in unliganded met forms at three of four globin subunits in the 24mer assembly and in oxygenated forms at the remaining A1 subunits. The partially unliganded structure showed remarkable structural changes at the AB loop regions causing quaternary rearrangements of the EF-dimer structure. In contrast, few changes occurred at the interface regions composed of the E and F helices. These results suggest that the ligand-induced structural changes of Oligobrachia Hb are quite different from those of the well-studied mollusk Hb having the same EF-dimer structure. The structural rearrangements make the dodecameric subassembly form a tighter conformation than those of fully oxygenated or CO-liganded dodecamer structure.     

Renal handling of phosphate, calcium, sodium, and potassium in intact and parathyroidectomized Rana pipiens

Renal excretion of phosphate, calcium, sodium, and potassium in intact and parathyroidectomized male Rana pipiens was studied by renal clearance techniques using 14C-inulin. In intact frogs, 57% of filtered phosphate, 60% of filtered calcium, 97% of filtered sodium, and 89% of filtered potassium was reabsorbed by the renal tubules. Following parathyroidectomy, the rate of reabsorption of phosphate became significantly higher than that of the intact frog, and the relative phosphate clearance (fractional excretion) decreased. These changes corresponded with a gradual rise in serum phosphate values. There was no major effect on excretion patterns of calcium, sodium, or potassium after parathyroidectomy. These results suggest that in frogs the parathyroid glands strongly influence phosphate excretion patterns but have little effect on the excretion of calcium, sodium, or potassium.     

Actual distribution of bacteriocytes in the trophosome of a beard worm (Oligobrachia mashikoi, Siboglinidae, Annelida): clarification using whole-mount in situ hybridization

Beard worms (Siboglinidae, Polychaeta) lack a mouth and a digestive tract and harbour chaemosynthetic bacteria in the bacteriocytes of the trophosome. Since beard worms depend on the organic compounds produced by the bacteria for nourishment, the bacteriocytes should be efficient in exchanging various substances with body fluids. For this reason, it is important to determine how the bacteriocytes are organized in the trophosome. As the first step of the present study, the appearance of bacteriocytes was examined in routinely stained paraffin sections. Second, visualization of the actual distribution of the bacteriocytes was attempted using whole‐mount in situ hybridization with a probe of the 16S rRNA nucleotide sequence of the bacterium. After routine haematoxylin & eosin staining, the bacteriocytes appeared to be aligned in cell cords accompanied with nutrient‐deposit cells that extended from both sides of the trophosome toward the dorsal side and folded up in the coelomic spaces. In whole‐mount preparations, however, bacteriocytes with intense signals of 16S rRNA were seen three‐dimensionally as many irregular leaves arranged from both sides of the ventral vessel toward the dorsal vessel. We will discuss the physiological significance of this characteristic distribution of the bacteriocytes in the present species.     

Biological Activities of p-Ethylphenyl and p-Acetylphenyl Phosphates and their Thiono Analogs

Sixteen phosphate or phosphorothioate esters related to neurotoxic tri-p-ethylphenyl phosphate and its active metabolites were synthesized and their biological activities including inhibitory activity against cholinesterases, insecticidal activity, toxicity to mammals and neurotoxicity were examined. Dialkyl p-ethylphenyl phoshates, p-acetylphenyl phosphates and their thiono analogs showed insecticidal activity, but did not show the ataxic sign by any sublethal doses in hens. When a methyl group was introduced on p-acetylphenyl ring, the biological activity changed remarkably by its position. The introduction of a methyl group into o-position made the ester inactive, while the introduction into m-position made it active to insects selectively.     

Prostaglandin E₂ increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E? (PGE?) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE?(10?? and 10?? M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE? (10?? to 10?? M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE? (10(-7) to 10?? M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE? treated-scales. Thus, PGE? acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE?-injencted goldfish increased significantly. We conclude that, in teleosts, PGE? activates both osteoblasts and osteoclasts and participates in calcium metabolism.