Complete nucleotide sequence of a new potexvirus, 'Cnidium virus X', isolated from Cnidium officinale in Japan.

A flexuous virus was detected in a Cnidium officinale plant in Japan showing mosaic symptoms. The virus was assigned to the genus Potexvirus based on analysis of its complete nucleotide sequence. The genomic RNA of the virus was 5,964 nucleotides in length, excluding the 3'-terminal poly(A) tail. It contained five open reading frames (ORFs), consistent with other members of Potexvirus. The ORF sequences differ from those of previously reported potexviruses. Phylogenetic analysis indicated that the polymerase of the virus is closely related to that of strawberry mild yellow edge virus; and the CP, to those of both yam virus X and vanilla virus X. We propose that this virus be designated as "cnidium virus X" (CnVX).

Morphological characteristics of olecranon fractures in adults : a Computed Tomography-based study.

The aim of this study was to identify the fragment's shape by evaluating olecranon fractures. We examined the CT images of 48 olecranon fractures (28 women and 20 men). Mean age was 59.9 years. On the olecranon's posterior surface, we measured the distance between the apex of the olecranon fragment and the radial edge of the flat spot on the short axis and the width of the flat spot on the same short axis. The tip radial ratio (i.e., the tip radial edge to the flat spot width) was derived from these parameters. The mean tip radial edge was 1.96 mm, and the flat spot width was 12.64 mm ; therefore, the tip radial ratio was 0.15 mm. Radial inclination on the articular surface was 30.55°. Our findings confirmed our hypothesis that the fracture lines run from the proximal ulnar side to the distal radial side on the olecranon's posterior and articular surfaces.

Regional spread of vanA- or vanB-positive Enterococcus gallinarum in hospitals and long-term care facilities in Kyoto prefecture, Japan.

Following an outbreak of vanA-positive Enterococcus faecium in 2005 in Kyoto prefecture, regional surveillance of vancomycin-resistant enterococci (VRE) was initiated. This revealed vanA- or vanB-positive Enterococcus gallinarum in multiple facilities. Eighty-eight vanA-positive E. gallinarum faecal carriers from 12 facilities and ten vanB-positive E. gallinarum faecal carriers from eight facilities were found. Pulsed-field gel electrophoresis profiles of the first isolate from each facility showed that 11 of the 12 vanA isolates and three of the eight vanB-positive E. gallinarum isolates belonged to a single clone. This study confirms the clonal spread of vanA- or vanB-positive E. gallinarum in a region and underlines the importance of surveillance of VRE for the presence of vancomycin resistance determinants.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

BACKGROUND: Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. OBJECTIVES: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. METHODS: We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. RESULTS: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. CONCLUSION: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Functional analysis of recombinant Bbeta15C and Bbeta15A fibrinogens demonstrates that Bbeta15G residue plays important roles in FPB release and in lateral aggregation of protofibrils.

BACKGROUND AND OBJECTIVES: Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant-variant fibrinogens. METHODS: We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala. RESULTS: Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys fibrinogen, functional analysis indicated (a) no thrombin-catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin- and reptilase-catalyzed fibrin polymerizations. For Bbeta15Ala fibrinogen, such analysis indicated slight impairments of both thrombin-catalyzed FPB release and lateral aggregation in thrombin-catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase-catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). CONCLUSION: We conclude that a region adjacent to Bbeta15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.

FSH suppression of nitric oxide synthesis in porcine oocytes.

The present study was designed to evaluate the regulation of nitric oxide (NO) synthesis in porcine oocytes during follicular development. Cumulus-oocyte complexes were obtained by aspirating the small follicles of immature porcine ovaries and cultured at 39 degrees C for 24-72 h with FSH in a serum-free medium. The oocyte-surrounding cumulus cells markedly proliferated and expressed LH receptor mRNA in response to FSH. The endothelial type of NO synthase (eNOS) (130 kDa) was detected in the oocyte, but not in the proliferated cumulus cells, by immunoblotting. The amount of oocyte eNOS did not significantly alter during culture, but measurement of nitrite and nitrate revealed FSH suppression of NO synthesis by approximately 50%. NO-releasing agents were added to the cultures to examine the effect of NO on the growth of cumulus cells. NO-releasing agents showed inhibitory effects on proliferation of the cumulus cells and expression of LH receptor mRNA. Thus, synthesis of eNOS-derived NO is suppressed in the porcine oocyte during development with no change in the enzyme amount, and it is suggested that it has an inhibitory function in the growth of cumulus cells.

Expression of endothelial nitric oxide synthase gene in cultured porcine granulosa cells after FSH stimulation.

The present study was designed to investigate nitric oxide (NO) synthesis and the expression of endothelial NO synthase (eNOS) gene in cultured porcine granulosa cells. Granulosa cells prepared from small follicles (1-4 mm diameter) were cultured in plastic dishes coated with fibronectin in chemically defined medium, and matured after 48 h of stimulation with FSH. The concentrations of nitrite and nitrate remained relatively constant until 42 h of stimulation, after which they increased significantly up to twofold at 48 h. NO synthesis was accompanied by an increase in cGMP. Gene expression for eNOS was studied by RT-PCR, and a PCR product of the expected size amplified. eNOS mRNA was expressed in the presence of FSH, but not in the absence of FSH. Although eNOS mRNA was not expressed in the initial period, it was expressed after 12 h of stimulation with FSH, and remained at a relatively constant level until 48 h. Expression of eNOS mRNA preceded expression of LH receptor mRNA, which showed a maximal level at 24 h of stimulation. These observations suggest that eNOS expression is not related to a rapid synthesis of NO in developing granulosa cells, and that the activation of NO synthesis is rigidly regulated in the initial period of development.

Inhibitory effect of retinoic acid on the development of immature porcine granulosa cells to mature cells.

The present study investigated the effect of retinoic acid (RA) on the differentiation of granulosa cells prepared from porcine ovaries. The granulosa cells were precultured for 15 h, then cultured for 48 h with FSH and further treated for 24 h with LH in order to induce their transformation into luteal cells. After the cells had been exposed to 1 microM retinoids (RA, retinal and retinol) for 87 h, analysis of the LH receptor mRNA expression, an indicator of granulosa cell differentiation, was carried out by using semiquantitative RT-PCR. The results showed that there was a decrease in LH receptor mRNA levels, and that RA had a more potent effect on these levels than the other two retinoids. When cells were exposed to RA in the immature stage (before the addition of FSH) or the early stage of development (0-24 h after the addition of FSH), expression of LH receptor mRNA was greatly diminished. When the immature cells were cultured for 15 h with RA, then washed and cultured for 48 h with FSH and for 24 h with LH, the expression of LH receptor mRNA was not reversed. In the differentiated cells (24 h after the addition of FSH), however, RA no longer had any inhibitory effect. When the immature cells were exposed to RA, FSH-induced expression of c-fos mRNA was markedly decreased. In contrast, expression of c-jun and activating transcription factor-4 mRNAs remained constant. However, the expression of c-fos mRNA was not decreased by forskolin. The results indicate that RA is a potent inhibitor in the immature stage of porcine granulosa cell differentiation, probably through decreased expression of FSH receptor, but that RA does not inhibit differentiation in the mature stage of the cells.

Repeated interferon-alpha administration inhibits dopaminergic neural activity in the mouse brain.

In vivo effects of single and repeated interferon-alpha administrations on the dynamics of noradrenaline, dopamine and 5-hydroxytryptamine were investigated in the mouse brain. Single interferon-alpha administration (15, 30 and 60 X 10(6) U/kg i.p.) had no significant effect on the levels of monoamines and their metabolites or monoamine turnover. When interferon-alpha (15 X 10(6) U/kg i.p.) was administered once a day for 5 days, however, both dopamine and 3,4-dihydroxyphenylacetic acid levels were significantly decreased and alpha-methyl-p-tyrosine-induced dopamine depletion was significantly suppressed. These results suggest that repeated interferon-alpha administration inhibits dopaminergic neural activity. This inhibitory action of interferon-alpha in dopamine neurons may be involved in adverse central effects, such as parkinsonism and depression with suicidal potential.

Critical role of nitric oxide in expression of porcine LH receptor at transcription and post-transcription levels.

The present study was performed to clarify the involvement of nitric oxide (NO) in the expression of LH receptor in porcine granulosa cells. The granulosa cells, prepared from porcine ovarian follicles, were developed in the presence of FSH. LH receptor mRNA was induced to reach a maximal level after a 24-h culture with FSH, as determined by semi-quantitative reverse transcriptase-PCR (RT-PCR). In our previous report (Nishida et al., 2000), we found that NO was released from granulosa cells after a 40-48 h culture with FSH. When 200 microM NO scavenger was added to cultures before the NO release (30 h), the content of LH receptor on the cells decreased to 28% that of the control. However, the receptor content was not influenced by addition of NO scavenger at 46 h, or by 50 microM NO donor at 30 or 46 h. During transformation of mature granulosa cells to luteal cells, LH receptor mRNA was induced after a 24-h culture with LH, which induction was inhibited by removal of endogenous NO. However, the expression was not influenced by addition of either NO scavenger at 46 h or by NO donor. During the period of these treatments, cellular DNA contents were constant. Consequently, the transient generation of NO may play a critical role in expression of the LH receptor at transcription and post-transcription levels.

Electrophysiological evidence for the typicality effect of human cognitive categorization.

Event-related brain potentials (ERPs) were recorded from 14 normal subjects during a category verification task. Stimulus words were selected from 17 semantic categories (e.g. 'vegetables'). Half of the words were typical category members (e.g. 'carrot', 'spinach') and the other half were atypical (e.g. 'parsley', 'asparagus'). Subjects were required to judge whether each stimulus belonged to a target category ('vegetables' or 'sports') or a non-target category. For the non-target category, the typicality effect was neither found in ERPs nor in reaction times. For the target category, typical words were responded to more quickly than were atypical words and the ERP amplitudes between a 300-450 ms period were more negative after the atypical words than after the typical words (typicality effect). These results suggested that typical words of the target were more primed by a target category than were the atypical words of the target and thus that a concept is represented by a prototype, the central tendency of all members of the category.

Production of chicken chimeras by fusing blastodermal cells with electroporation.

AIM: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cells fused with electroporation. METHODS: Stage-X blastodermal cells isolated from freshly laid fertile unincubated white Leghorn and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferred to the recovery medium (DMEM containing 10% FBS) and was injected into the subgerminal cavity of recipient unincubated embryos (stage X). RESULTS: Of 177 recipient embryos injected with the fusing blastodermal cells, 6 (3.4%) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence of feathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days of incubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromosome-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other exhibited the same DNA in the left and right gonads and other tissues, but not the stomach. CONCLUSION: Recipient embryo having electrofused blastodermal cells yields somatic and germline chimeric chickens more successfully.

Proliferation of exogenously injected primordial germ cells (PGCs) into busulfan-treated chicken embryos.

AIM: This study was designed to investigate the effect of busulfan treatment on the proliferation of chicken primordial germ cells (PGCs) in vivo, focusing on the preferential settlement of PGCs onto the germinal ridges of chicken embryos. METHODS: Busulfan (250 ng/egg) was injected into the egg white of freshly oviposited fertilized eggs, which were then incubated. Embryonic development and viability were examined, and exogenous PGCs collected from embryonic blood vessels were injected into the germinal crescent region of recipient embryos. The number of PGCs resided onto germinal ridges of the right and left sides were compared. RESULTS: Busulfan had a slight harmful effect on the embryo viability and the PGCs proliferation. The number of PGCs resided onto the left side of germinal ridges was slightly higher as compared with the right side. CONCLUSION: Busulfan suppressed the viability of embryos and the proliferation of endogenous PGCs in the recipient embryos. However, the number of exogenous PGCs proliferated was higher in embryos treated with busulfan than those without busulfan. Data also suggest the possibility of a preferential residence of PGCs toward the left side of the germinal crescent region as compared with the right, which may be due to a more advanced functional development of the left gonad than the right.

Reproductive characteristics of transgenic (TG) chickens carrying an exogenous gene.

An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was confirmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chicks were raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female transgenic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination according to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the embryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As a result, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts from embryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of injected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated successfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens.

Effect of age on quality of fresh and frozen-thawed semen in White Italian ganders.

AIM: To comparatively evaluate the fresh semen quality of 1, 2 and 3-yr-old White Italian ganders (Anser anser L.) and the susceptibility of spermatozoa to freezing-thawing procedure. METHODS: Semen was collected by dorso-abdominal massage every 2 days~3 days from three groups of ganders: 1-yr-old (n=11), 2-yr-old (n=7) and 3-yr-old (n=9). In the pooled fresh semen samples, the following parameters were evaluated: the ejaculate volume, the blood or feca contamination and the motility, concentration and morphology of spermatozoa. Sperm motility and morphology were evaluated in the frozen-thawed semen. Semen diluted with EK extender was frozen in straws in a computerized freezing unit with 6 % dimethyl-formamide to -140 deg at a rate 60 deg/min and then transferred into the LN2 container. Straws with semen were thawed in a water bath at 60 deg. RESULTS: The ejaculate volume decreased with the age (0.21 mL for 1-yr-old, 0.18 mL for 2-yr-old and 0.14 mL for 3-yr-old ganders); the sperm concentration increased with the age (327 x 10(6) mL(-1) for 1-yr-old, 431 x 10(6) mL(-1) for 2-yr-old and 547x10(6) mL(-1) for 3-yr-old ganders); the number of live - normal sperm was significantly (P<0.01) lower in the 1-yr-old than that in the 2- and 3-yr-old ganders (26.61 %, 41.54 and 35.9 %, respectively). The percentage of normal cells survived the freezing-thawing process was 37.7 %, 43.3 % and 40.9 % for 1-, 2- and 3-yr-old ganders, respectively. CONCLUSION: Freezing and thawing processes more significantly (P<0.01) affected the motility, viability and morphology of spermatozoa in semen of 1-yr-old ganders in comparison with older males.

Comparative study on semen quality of one- and two-year-old ganders during the entire reproductive season.

AIM: To evaluate the characteristics of semen produced by one- and two-years old White Italian ganders during the entire reproductive season, in order to clarify whether the young ganders are responsible for a low fertility rate in young geese. METHODS: Males were kept individually in cages under natural light. Semen was collected by dorso-abdominal massage three times a week and routine examination was performed. RESULTS: The mean ejaculate volume (2.1 and 1.6 mL, respectively) and sperm concentration (323 and 281 x 10(6)/mL, respectively) in one-year-old ganders were higher than those of two-year-old ones. The percentages viable spermatozoa of one- and two-year-old ganders were similar (91.4 and 92.3%, respectively), but the percentage of normally formed viable spermatozoa was significantly higher in the older ganders than in the younger (47.8 and 42.9%, respectively, P < 0.05). CONCLUSION: The semina from one- or two-year-old Ganders were similar in regard to volume, sperm density and sperm motility, but the percentage of normally formed viable spermatozoa, which is critical for fertilization, was significantly higher in the older ganders. It appears that the ganders are responsible for the low fertility rate in young geese.

Introduction of DT40 cells into chick embryos.

AIM: To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avian leukosis virus (ALV)-induced chicken B cell line DT40 to the production of chimeric birds. METHODS: The DT40 cells incorporated with exogenous gene ( lacZ constructs encoding Escherichia coli beta-galactosidase: beta-gal) were introduced into chick embryos by the injection of cells into stage X blastoderm. Manipulated eggs were incubated for 3 (trial 1) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of beta-galactosidase and polymerase chain reaction (PCR) analysis. RESULTS: The survival rates of the manipulated embryos incubated for 3 days (stage 18-20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively. The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, for the survived embryos. CONCLUSION: The rate of embryonic viability and expression rate of introduced genes were not so high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chick embryos.

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts.

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.

Effect on turkey spermatozoa of a frothy fluid derived from the cloaca of a male turkey.

The influence on turkey spermatozoa of a frothy fluid derived from the cloacal region of a male turkey was investigated. The frothy fluid was collected from the turkey tom during mounting, and semen for the experiment was obtained from the ductus deferens removed after necropsy. Spermatozoa diluted with frothy fluid were examined for motility, viability, and fertilizing capacity and compared with semen diluted with phosphate buffer or undiluted ductal semen. The life-span of spermatozoa suspended in frothy fluid was slightly prolonged during in vitro storage as compared with the undiluted semen or the semen diluted with phosphate buffer; however, a rapid increase of the number of deformed spermatozoa during storage was observed in the semen diluted with frothy fluid. The fertilizing ability of spermatozoa was not influenced by the dilution with frothy fluid when the diluted spermatozoa were inseminated intravaginally immediately after the dilution. On the contrary, when spermatozoa suspended in frothy fluid were preserved at 0 C for 24 h, their fertilizing capacity decreased drastically, probably due to the increased number of abnormal spermatozoa during in vitro preservation.

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.