Ecology,physiology and genetics of a phycodnavirus infecting the noxious bloom-forming raphidophyte <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis>

Abstract:   Heterosigma akashiwo virus (HaV) is a large icosahedral virus (∼0.2 μm) harboring a double-stranded DNA (dsDNA) genome (∼294 kbp). The virus is the only member of the genus Raphidovirus in the family Phycodnaviridae. Since its first discovery, a number of ecologic, physiologic and genetic studies about HaV have been conducted; especially, the relationship between H. akashiwo and HaV in nature was studied and viral infection is now regarded as a significant factor influencing the dynamics and termination of H. akashiwo blooms. HaV infection has considerable impacts on H. akashiwo populations in both aspects of fluctuation in biomass (quantity) and changes in clonal composition (quality). Partial sequencing of the HaV genome revealed that a number of genes showed considerable similarity to those of other protist-infecting viruses; still, the phylogenetic position of HaV suggested a number of enigmas in host–virus coevolution. Here are summarized the ecology, physiology and genetics of HaV especially from the viewpoint of the host–virus relationship.     

Chemical structures of the condensed tannins in the fruits of Diospyros species

The structural variety of the condensed tannins (proanthocyanidins) in the fruits of 16Diospyros species are reported. Eleven species contained condensed tannins mostly consisting of a mixture of catechin (CA) and gallocatechin (GCA) repeating units; the other five species did not. The GCA content in the CA-GCA total varied from 0.3% to 84.6%. The number of esterified gallic acid per one flavan repeating unit (degree of galloylation, DG) ranged from 0.01 to 0.89. The GCA content was found to be proportional to the DG values. Thus, 16Diospyros species tested may be classified into five groups by the analytical data of their condensed tannins. It may be interesting to compare their structural characteristics with those of the condensed tannins in other fruits, leaves, woods, and barks from the viewpoint of their biosynthesis and function in the plants.Part of this paper was presented at the 46th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1995     

Responses to the Standard for Exchange of Nonclinical Data (SEND) in non-US countries

The Standard for the Exchange of Nonclinical Data (SEND), adopted by the US FDA, is part of a set of regulations and guidances requiring the submission of standardized electronic study data for nonclinical and clinical data submissions. SEND is the nonclinical implementation of SDTM (Study Data Tabulation Model), the standard electronic format for clinical regulatory submissions to FDA. SEND, SDTM, and the associated Controlled Terminology have been developed by CDISC (Clinical Data Interchange Standards Consortium). In order to successfully implement SEND, interdisciplinary contributions between sponsors and CROs, need a model for task allocation. This is being undertaken by the Pharmaceutical Users Software Exchange (PhUSE). Because SEND is currently the preferred submission format of the US FDA only and will become required by it starting in December 2016, only American academic societies and companies are actively involved. An exception to this is the INHAND initiative, which leads the way in standardizing terminology for toxicological pathology. On the other hand, international globalization of other clinical and nonclinical practices is not feasible because there are substantial differences between the US and non-US countries in CRO involvement in drug development. Thus, non-US countries must consider and develop approaches to SEND that meet their needs. This paper summarizes the activities of the major organizations involved in SEND development and implementation, discusses the effective use of SEND, and details a compliance scheme (research material of the Showa University School of Medicine) illustrating how pharmaceutical companies can complete a large amount of work up to an FDA application with the effective utilization of CROs and solution providers.     

Nitrogen-fixing activity in decomposing litter of three tree species at a watershed in eastern Japan

Nitrogen fixation during litter decomposition was studied for 34 months using litterbags containing newly fallen litter of coniferous species Cryptomeria japonica and Pinus densiflora and that of deciduous species Quercus serrata. Litterbags were set in contact with the forest floor in a deciduous broad-leaved forest near the top of a slope and in a C. japonica stand at the middle of the slope at a watershed in eastern Japan. Nitrogen-fixing activity, estimated by acetylene reduction after 16 and 19 months of incubation, was 62.65–3.86 nmoles C₂H₄ h⁻¹ g⁻¹ DW in Cryptomeria litter, but only 1.07–0.09 in Pinus and 0.72–0.04 in Quercus. The rate of N increase in decomposing litter was highest in Cryptomeria. Fungal biomass in decomposing litter, estimated by ergosterol content, increased during the initial 16 months of incubation in Cryptomeria and Quercus, and during the initial 19 months of incubation in Pinus. The litter decomposition rate was highest in Cryptomeria among the three species, due to increased N content and fungal biomass in Cryptomeria litter. Thus, N increase in decomposing Cryptomeria litter affects the subsequent N dynamics and decomposition pattern.     

Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis

Cathepsin K was originally identified as an osteoclast-specific lysosomal protease, the inhibitor of which has been considered might have therapeutic potential. We show that inhibition of cathepsin K could potently suppress autoimmune inflammation of the joints as well as osteoclastic bone resorption in autoimmune arthritis. Furthermore, cathepsin K-/- mice were resistant to experimental autoimmune encephalomyelitis. Pharmacological inhibition or targeted disruption of cathepsin K resulted in defective Toll-like receptor 9 signaling in dendritic cells in response to unmethylated CpG DNA, which in turn led to attenuated induction of T helper 17 cells, without affecting the antigen-presenting ability of dendritic cells. These results suggest that cathepsin K plays an important role in the immune system and may serve as a valid therapeutic target in autoimmune diseases.     

Externally suppressible proline quadruplet ccc U

Three (+1) frameshift mutations located at different genetic sites respond with high specificity to the same external suppressor. In each case, the suppressor restores small amounts of protein that is normal in electrophoretic mobility and heat stability. One of these proteins has been shown to have the wildtype amino acid sequence. The messenger RNA quadruplet CCCUappears to be common to all three frameshift sites and to be translated by the suppressor as proline. A likely suppressor agent is a proline transfer RNA with a quadruplet anticodon or its functional equivalent.     

Comparison of estrus induction and subsequent fertility with two different intravaginal devices in ewes during the non-breeding season

Two experiments were conducted to compare the effect of estrus induction by controlled internal drug release (CIDR) and intravaginal cream containing 500 mg progesterone (P cream) in ewes during the non-breeding season. In the first experiment, twenty-four ewes were randomly grouped for two treatments with the different intravaginal devices for 12 days: Group A was the CIDR group and Group B was the P cream group. Blood was collected from all treated ewes, and progesterone (P(4)), estradiol 17-beta (E(2)) and luteinizing hormone (LH) concentrations were measured by enzyme immunoassay. In the second experiment, the conception rates from natural mating, estrus-detected AI (inseminated 12 h after estrus detection), or fixed-time AI (inseminated 42 h after removal of an intravaginal device) in 127 ewes treated with CIDR or P cream were compared. In Experiment 1, the rate of estrus induction and the time of estrus onset after device removal were 91.7% and 36.3 +/- 15.7 h in Group A, and 100% and 35.0 +/- 12.6 h in Group B, respectively. There were no significant differences between the devices. The mean plasma P(4) concentration in Group B was significantly (P < 0.01) lower than Group A between day -9 and day -1 (Day 0: the day of device removal). However, no significant differences were found in the mean E(2) concentrations of the two groups after treatment. The mean time of estrus onset in ewes with an observed LH surge and the time of LH surge after treatment were 23.3 +/- 8.7 h and 30.3 +/- 5.0 h for Group A and 27.6 +/- 6.5 and 26.3 +/- 8.0 h for Group B, respectively, and there were no significant differences. However, a significant difference (P < 0.05) was found in the mean time from the time of estrus onset to LH surge between Group A (6.4 +/- 6.7 h) and Group B (-1.3 +/- 4.1 h). In Experiment 2, the conception rates for natural mating, estrus-detected AI, and fixed-time AI were 55.0, 29.4, and 25.0% for Group A and 40.7, 25.0, and 42.1% for Group B, respectively, and there were no significant differences. These results suggest that the effect of induction of estrus and ovulation and the rate of conception after treatment were comparable to CIDR even though the plasma P(4) concentration of the P cream method tended to be low during the insertion period.     

Prevalence of Borna disease virus antibodies in healthy Japanese black cattle in Kyushu

Epidemiological studies have demonstrated that asymptomatic infection of Borna disease virus (BDV) is found in various species of animals in Japan. Recent reports have also revealed that neurological diseases caused by this virus could exist in horses, cattle, a dog, and cats in this country. In this study, we investigated seroprevalence of BDV antibodies in Japanese black cows reared in Kyushu, the southernmost main island of Japan, using ELISA and Western-immunoblotting. Of 101 serum samples, 11 (10.9%) and 21(20.7%) sera were identified as having antibodies to the BDV N and P antigens, respectively. Among the positive sera, three cows (2.9%) were seropositive for both of the antigens. Furthermore, interestingly, only female cows showed antibodies to P, whereas N antibodies were detected in male and female cows with a comparative ratio. Together with previous studies, our results indicate that BDV might be widely spread in cattle raised in Japan. Furthermore, this is the first report to show that beef cattle, Japanese black cattle, have antibodies against a possible zoonotic pathogen, BDV.     

Effects of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram spermatozoa

In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 +/- 2.9 and 58.3 +/- 6.7%, respectively) than in the positive control (F) group (41.7 +/- 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 +/- 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 +/- 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 +/- 1.3 and 6.3 +/- 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 +/- 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 +/- 2.4 and 25.0 +/- 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 +/- 2.4 and 18.8 +/- 1.3%, respectively) and F (28.8 +/- 3.8 and 18.8 +/- 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing.     

Changes of fructose concentrations in seminal plasma and glucose and testosterone concentrations in blood plasma in rams over the course of a year

The present study was performed to examine seasonal changes in the fructose concentrations of seminal plasma and glucose and testosterone concentrations of blood plasma over the course of a year (from November 2004 to November 2005) using 5 Suffolk rams. Osmolality of the seminal plasma was also measured. The fructose concentrations in the seminal plasma increased as the breeding season approached, with the maximum in October (179.8 mg/dl) and the minimum in May (6.9 mg/dl), although there were no significant differences during the year. Osmolality of the seminal plasma in February (304 mOsm) was significantly (P<0.05) lower than in January (325 mOsm), July (327 mOsm), and August (325 mOsm). It was also significantly (P<0.05) lower in November (308 mOsm) than in January and August. The blood plasma glucose concentration in October (79.3 mg/dl) was significantly (P<0.05) higher than in January and February (43.2 and 43.7 mg/dl, respectively). The blood plasma testosterone (T) concentrations were significantly (P<0.05) higher in September (8.5 ng/ml) and October (10.2 ng/ml) than in other months. The fructose concentrations in the seminal plasma appeared to be related to the glucose and T concentrations in the blood plasma. These results show that fructose concentrations in the seminal plasma and blood plasma glucose and T concentrations tended to increase during the breeding season, with the highest concentrations in October.