Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

Stochastic variation of system component variables in a network analysis

A deterministic network simulation was reformulated to include the uncertainty of various parameters. Then, by the recording values obtained for each pathway or system in the network for a set of stochastic parameters and then choosing another set of stochastic parameters and recording the values obtained from that simulation, an average value and standard deviation for any system in the network can be determined. In the decision-making process of choosing from one system over another, the manager will have a better understanding of the complex interactions involved by knowing the effects that uncertainty in the system can cause.     

Three cases of mare sterility with sex-chromosomal abnormality (63, X)

Contents: Three mares that had heen diagnozed clinically as sterile were cytogenetically investigated. For each o f the mures 63 or more metaphases were analyzed (63 metaphases in one case, 72 metaphases in the 2nd case and 90 rnetaphases in the 3rd case). Only the sex chromosomal monosomy (63, X) were detected in each of the metaphases. Using the G-banding technique unpairing X-chromosomes, was accurately distinguished from autosomes nos. 1 and 2. The incidence of drumstick was 0.5 76 and 0.2 % respectively in two cases. The most obvious clinical abnormality was gonadal dysgenesis, which was observed in all mares. N o clear-cut oestrus symptoms were detectable despite repeated treatment with various hormones.     

Dental incremental lines in Sika deer (Cervus nippon); polarized light and fluorescence microscopy of ground sections

Periodic growth incremental lines are found universally in dental hard tissues. This periodicity theoretically allows for estimation of age, even in days, which would be useful in studies of wild animals. In the present study, enamel and dentin increments of the sika deer (Cervus nippon) were observed in ground sections with a polarized light microscope, and their periodicity was examined by the use of a chronological labeling method with fluorochromes. Enamel increments occurred at a mean interval of 10.6 (SD=1.5) microm, and mean spacing of dentin increments was 17.3 (SD=1.8) microm. Fluorochromic marking revealed that incremental lines form each day in enamel and almost every second day in dentin. The fluorescence-labeled lines suggest that enamel formation of the first molar is complete by the age of 5 months. Due to its longer interval of incremental lines and longer term of formation, we conclude that dentin is more suitable than enamel for day-age estimation in sika deer. Experimental confirmation of incremental growth periodicity in various species can improve the reliability of use of tooth increments for age estimation and life history reconstruction.     

Molecular ordering of organic molten salts triggered by single-walled carbon nanotubes

When mixed with imidazolium ion-based room-temperature ionic liquid, pristine single-walled carbon nanotubes formed gels after being ground. The heavily entangled nanotube bundles were found to untangle within the gel to form much finer bundles. Phase transition and rheological properties suggest that the gels are formed by physical cross-linking of the nanotube bundles, mediated by local molecular ordering of the ionic liquids rather than by entanglement of the nanotubes. The gels were thermally stable and did not shrivel, even under reduced pressure resulting from the nonvolatility of the ionic liquids, but they would readily undergo a gel-to-solid transition on absorbent materials. The use of a polymerizable ionic liquid as the gelling medium allows for the fabrication of a highly electroconductive polymer/nanotube composite material, which showed a substantial enhancement in dynamic hardness.     

Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock

Mice lacking mCry1 and mCry2 are behaviorally arrhythmic. As shown here, cyclic expression of the clock genes mPer1 and mPer2 (mammalian Period genes 1 and 2) in the suprachiasmatic nucleus and peripheral tissues is abolished and mPer1 and mPer2 mRNA levels are constitutively high. These findings indicate that the biological clock is eliminated in the absence of both mCRY1 and mCRY2 (mammalian cryptochromes 1 and 2) and support the idea that mammalian CRY proteins act in the negative limb of the circadian feedback loop. The mCry double-mutant mice retain the ability to have mPer1 and mPer2 expression induced by a brief light stimulus known to phase-shift the biological clock in wild-type animals. Thus, mCRY1 and mCRY2 are dispensable for light-induced phase shifting of the biological clock.     

Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.