Molecular-dynamics simulations of [Met5]- and [D-Ala2,Met5]-enkephalins. Biological implication of monomeric folded and dimeric unfolded conformations.

To investigate the biologically active conformation of enkephalin, molecular-dynamics simulations were applied to [Met5]- and [D-Ala2,Met5]-enkephalins. The dynamic trajectory of monomeric extended [Met5]-enkephalin was analysed in terms of relative mobility between respective torsions of backbone chain. After 10 ps of the dynamics simulation, the conformational transition was converged into a stationary state among the beta-bend folded forms, where they are stabilized by several intramolecular hydrogen-bond formations. Similar conformational transition was also observed in the dynamics simulation of [D-Ala2,Met5]enkephalin, which is a more mu-receptor-specific peptide than [Met5]enkephalin. The geometrical correspondence between the monomeric enkephalin conformation in the stationary state and morphine molecule (a mu-specific rigid opiate) was surveyed by virtue of the triangular substructures generated by choosing three functional atoms in each molecule, and good resemblances were observed. On the other hand, the dynamics simulation of the antiparallel extended [Met5]enkephalin dimer showed a trajectory different from that of the monomeric one. Two intermolecular hydrogen bonds at Tyr1 (NH3+)...Met5(CO2-) end residues were held throughout the 100 ps simulation, the dimeric structure being consequently kept. The conformational transition of the backbone chains from the antiparallel extended form to the twisted one took place via an intermediate state. Many conformations revealed during the dynamics simulation showed that the relative orientations of each two Tyr1, Gly3, Phe4 and Met5 residues in the dimer are nearly related by a pseudo-C2-symmetry respectively, and both halves of the dimer structure could be further fitted to the monomeric folded enkephalin conformation. The monomeric and dimeric conformations of enkephalin at their stationary states are discussed in relation to the substrate-specificity for mu- and delta-opioid receptors.     

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR^H) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR^H by the standard phosphoramidite solid-phase method, dR^H was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR^H at their 3′-overhang regions were synthesized. Introducing dR^H to the 3′-end of the antisense strand of siRNA reduced its knockdown effect.     

Evaluation of Chlorella as a Decorporation Agent to Enhance the Elimination of Radioactive Strontium from Body

Background

Release of radionuclides, such as ¹³⁷Cs and ⁹⁰Sr, into the atmosphere and the ocean presents an important problem because internal exposure to ¹³⁷Cs and ⁹⁰Sr could be very harmful to humans. Chlorella has been reported to be effective in enhancing the excretion of heavy metals; thus, we hypothesized that Chlorella could also enhance the elimination of ¹³⁷Cs or ⁹⁰Sr from the body. We evaluated the potential of Chlorella as a decorporation agent in vitro and in vivo, using ⁸⁵Sr instead of ⁹⁰Sr.

Methods

In vitro experiments of adsorption of ¹³⁷Cs and ⁸⁵Sr to Chlorella were performed under wide pH conditions. The maximum sorption capacity of Chlorella to strontium was estimated using the Langmuir model. A ⁸⁵Sr solution was orally administrated to mice pretreated with Chlorella. At 48 h after ⁸⁵Sr administration, the biodistribution of radioactivity was determined.

Results

In the in vitro experiments, although ⁸⁵Sr barely adsorbed to Chlorella at low pH, the ⁸⁵Sr adsorption ratio to Chlorella increased with increasing pH. The maximum sorption capacity of Chlorella to strontium was 9.06 mg / g. ¹³⁷Cs barely adsorbed to Chlorella under any pH conditions. In the biodistribution experiments, bone accumulation of radioactivity after ⁸⁵Sr administration was significantly decreased in the Chlorella pretreatment group compared with the non-treatment control group.

Conclusions

In conclusion, these results indicated that Chlorella could inhibit the absorption of ⁹⁰Sr into the blood and enhance the elimination of ⁹⁰Sr from the body through adsorption in intestine. Further studies are required to elucidate the mechanism and the components of Chlorella needed for adsorption to strontium and could promote the development of more effective decorporation agents.     

Adult-type pigment cells,which color the ocular sides of flounders at metamorphosis,localize as precursor cells at the proximal parts of the dorsal and anal fins in early larvae

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.     

Müllerian carcinofibroma of the uterus. A case report.

BACKGROUND: Müllerian carcinofibroma is composed of malignant epithelial tumor (cancer) and benign mesenchymal tumors. It is the least frequent among mixed müllerian tumors. There are eight reported cases of carcinofibroma or cases showing similar histology, with only two of these cases recurrent. CASE: A case of müllerian carcinofibroma arose in the uterine body. The patient was an 83-year-old, postmenopausal female whose endometrial cytology revealed cell clusters of adenocarcinoma and scattered nonepithelial cells with enlarged nuclei without nuclear atypism or mitosis. Histology of the resected uterus showed a mixture of well to poorly differentiated adenocarcinoma, and fibromatous and leiomyomatous nonepithelial tumors without a transition between them. There was no sign of recurrence nine months after hysterectomy. CONCLUSION: Müllerian carcinofibroma seems to have a better prognosis than malignant mixed müllerian tumor. When both cancer cells and an abundance of nonepithelial cells are seen on gynecologic cytology, it may be important to consider mixed müllerian tumor and to differentiate müllerian carcinofibroma from malignant mixed Müllerian tumor by careful observation of the nuclear size, nucleoli, nuclear atypism and mitosis of the nonepithelial cells.     

Effect of genetically defined oocyte depletion on production of androgens and oestrogens by ovaries of suckling mice

Steroid production and histological features of ovaries were compared either among normal +/+ mice of 3-12 days of age or among 12-day old mutant mice with various degrees of oocyte depletion. Whole ovaries were cultured in the medium containing [3H]progesterone and hCG or 4-androstene-3,17-dione and FSH; amounts of [3H]androgens or oestrogens released from the ovaries were assayed. FSH-responsive aromatase activity was detectable in ovaries of +/+ mice on day 3 after birth (2.6 +/- 0.4 pmol/2 ovaries/48 h), but the activity producing androgens from progesterone, under stimulation of hCG, was not detectable even on day 6 after birth (less than 0.1 pmol/2 ovaries/48 h). The androgen-producing activity appeared on day 9 after birth (1.16 +/- 0.25 pmol/2 ovaries/48 h), when follicles with more than two layers of granulosa cells developed. The ovaries of 12-day old Sl/Slt mice contained a considerable number of follicles with a single layer of granulosa cells, but did not contain any follicles with more than two layers of granulosa cells. The ovaries of Sl/Slt mice possessed aromatase activity (3.3 +/- 0.4 pmol/2 ovaries/48 h) but, not androgen-producing activity (less than 0.1 pmol/2 ovaries/48 h). The present results suggest that development of follicles with more than two layers of granulosa cells may induce the activity producing androgens from progesterone under stimulation of LH in suckling mouse ovaries, though the FSH-responsive aromatase activity is present even in follicles with a single layer of granulosa cells.     

Range-wide genetic structure in a north-east Asian spruce (Picea jezoensis) determined using nuclear microsatellite markers

Aim We used microsatellite markers to determine the range‐wide genetic structure of Picea jezoensis and to test the hypothesis that the past population history of this widespread cold‐temperate spruce has resulted in a low level of genetic variation and in imprints of inbreeding and bottlenecks in isolated marginal populations. Location The natural range of the three infraspecific taxa of P. jezoensis throughout north‐east Asia, including isolated marginal populations. Methods We analysed a total of 990 individuals across 33 natural populations using four nuclear microsatellite loci. Population genetic structure was assessed by analysing genetic diversity indices for each population, examining clustering (model‐based and distance‐based) among populations, evaluating signals of recent bottlenecks, and testing for isolation by distance (IBD). Results The 33 populations were clustered into five groups. The isolated marginal groups of populations (in Kamchatka, Kii in Japan and South Korea) exhibited low levels of allelic richness and gene diversity and a complete or almost complete loss of rare alleles. A recent bottleneck was detected in the populations in Hokkaido across to mid‐Sakhalin. The IBD analysis revealed that genetic divergence between populations was higher for populations separated by straits. Main conclusions Picea jezoensis showed a higher level of genetic differentiation among populations (F_ST = 0.101) than that observed in the genus Picea in general. This might be attributable to the fact that historically the straits around Japan acted as barriers to the movement of seeds and pollen. The low levels of genetic diversity in the isolated marginal population groups may reflect genetic drift that has occurred after isolation. Evidence of a significant bottleneck between the Hokkaido and mid‐Sakhalin populations implies that the cold, dry climate in the late Pleistocene resulted in the decline and contraction of populations, and that there was a subsequent expansion followed by a founder effect when conditions improved. The high polymorphism observed in P. jezoensis nuclear microsatellites revealed cryptic genetic structure that organellar DNA markers failed to identify in a previous study.