The effect of heating against Cryptosporidium oocysts

The effect of heat treatment was examined against oocysts of Cryptosporidium parvum, Cryptosporidium muris and chicken Cryptosporidium sp. isolated in Japan. The oocysts of these species were exposed at 50, 55, 60 and 70 degrees C for 5, 15, 30 and 60 sec in water bath, respectively. To determine the infectivity of heated oocysts, the nice and chickens were inoculated with the treated oocysts and the oocyst output in the feces after inoculation was examined. In C. parvum and chicken Cryptosporidium sp., the oocysts were not detected from mice or chickens which were received oocysts heated at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. In C. muris, the oocysts were not detected from mice which were received oocysts heated at 55 degrees C for 15 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec. Consequently, it was clarified that the infectivity of Cryptosporidium oocysts to mice and chickens was lost by heating at 55 degrees C for 30 sec, 60 degrees C for 15 sec and 70 degrees C for 5 sec.     

Somatosensory evoked potentials produced by stimulation of the dorsomedial nerves innervating the tail in dogs

To record the somatosensory evoked potentials (SEPs) produced by stimulation of tail nerves and determine the effects of acute compression of the cauda equina on SEPs. The subjects were 10 adult Beagles. SEPs were recorded after stimulating the dorsomedial nerves (DMN) innervating the tail. The cauda equina was compressed using a balloon catheter inserted into the vertebral arch. In SEPs, two negative and one positive peak were often observed. The compression of the cauda equina caused significant depression of the positive component. The SEPs produced by stimulation of the DMN reflect the activities of ascending neuronal pathways above the coccygeal spinal segments and may be a useful tool for examining cauda equina syndrome.     

Cellular architecture of the synovium in the tendon sheath of horses: an immunohistochemical and scanning electron microscopic study

The intimal lining cells of the synovium in joints have been studied morphologically and histochemically and shown to consist of macrophagic cells (type A) and fibroblast-like cells (type B). It is believed that the structure of the synovium in the tendon sheath is similar to that in the joint, but there have been only a few morphological studies of the tendon sheath. The present study revealed the cellular architecture of synovium in the tendon sheath of horses by histochemistry and scanning electron microscopy (SEM). Like the joint, the inner surface of the tendon sheath was covered with a cell-rich intimal layer. Acid phosphatase-positive A cells accumulated in the mesotendon but few in other regions. B cells were selectively immunolabeled with protein gene product (PGP) 9.5 antiserum and distributed in the entire length of the synovial intima in the tendon sheath. The synovial intima consisted of a surface layer rich in the processes of B cells and a deep layer containing cell bodies of B cells. Using SEM, B cells could be classified into two types according to the morphology of their processes. B cells of dendritic type were located mainly in the joint-side of the tendon sheath and extended branched processes to form a meshwork on the intimal surface. B cells of flat type were located in the skin-side of the tendon sheath and in the mesotendon. Their membranous processes extended in a horizontal direction and covered the intimal surface, resembling epithelium. It appears likely that the morphology and distribution of synovial intimal cells are influenced by various factors, such as the nature of the underlying tissues and the magnitude of mechanical stress.     

Laccase Gene LAC1 of Colletotrichum lagenarium Is Not Essential for Melanin Biosynthesis and Pathogenicity

Laccase has been shown to oxidize 1,8-dihydroxynaphthalene (1,8-DHN) in the final step of melanin biosynthesis in several fungi. In this study, a laccase gene (LAC1) was cloned from Colletotrichum lagenarium that synthesizes 1,8-DHN melanin, and characterized. To clone the LAC1 sequences, genomic DNA was subject to polymerase chain reactions (PCR) with degenerate oligonucleotide primers that were designed on the basis of amino acid sequences conserved among characterized laccases from other ascomycetes Botrytis cinerea, Neurospora crassa, Aspergillus nidulans, and Cryphonectria parasiticus. The LAC1 gene contained an open reading frame composed of 589 codons and three introns of 51, 49, and 57 nucleotides. The deduced amino acid sequence of Lac1p had high similarity to that of laccase from N. crassa and significant homology with those of multicopper blue proteins. Under melanin-induced culture of this fungus, laccase activity significantly increased and LAC1 expression was also detected. However, the lac1Δ mutants retained laccase activity and had no significant phenotypic differences in melanin production or pathogenicity from the wild-type strain. Received 23 February 2001/ Accepted in revised form 29 March 2001     

Bacteriocin production of probiotic Lactobacillus gasseri LA39 isolated from human feces in milk-based media

The use of bacteriocins from Lactobacillus gasseri , a probiotic lactic acid bacterium, as bio-preservatives in the food industry and animal formulations has been limited because few strains of Lb. gasseri are cultivated and produce a bacteriocin in natural media such as milk and milk-based media. By the determination of the growth-supplements to milk among the 47 nutrients, Lb. gasseri JCM1131^T, LA39 and LA158 isolated from human feces were successfully cultured in reconstituted skim milk and cheese whey using proteose peptone as a nutrient supplement, where Lb. gasseri LA39 produced a useful bacteriocin, gassericin A, with effective growth-inhibiting activity against Gram-positive food-borne pathogens. The data suggest these developed low-cost safe media supporting enough production of bacteriocins by the probiotic Lb. gasseri LA39 could be used to improve the safe bio-preservation of foods and therapy of bovine mastitis, and extra cheese whey produced by cheese making industry is reused in the cultivation for probiotics effectively.     

Effect of centrifugation treatment before vitrification on the viability of porcine mature oocytes and zygotes produced in vitro

The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 x g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes.     

The luteotrophic function of galectin-1 by binding to the glycans on vascular endothelial growth factor receptor-2 in bovine luteal cells

The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.     

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars,combinations of permeating cryoprotectants and equilibration regimens

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5–15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.     

Nitrogen promotes water consumption in seedlings of Cryptomeria japonica but not in Chamaecyparis obtusa

To evaluate the influence of excessive N deposition on the water consumption of a Japanese plantation forest, 1-year-old seedlings of major plantation trees, Cryptomeria japonica (Japanese cedar) and Chamaecyparis obtusa (Hinoki cypress), were treated with combinations of two N levels (Moderate N and High N) and two soil water conditions (Dry and Moist) for 4 months. The High N treatment received five times as much N as in the Moderate N treatment; the total amount of N added in the High N treatment was roughly 25 times the annual N deposition in precipitation. An increase in soil N availability increased the needle transpiration rate, needle biomass, and needle N content of C. japonica under the Moist treatment, whereas those of C. obtusa were not significantly affected by soil N treatment at either soil water level. Needle N content in C. japonica was positively related to needle photosynthetic rate and transpiration rate. Our results suggested that excessive N deposition has the potential to enhance water consumption in C. japonica stands on moist soils. However, the effects of increased N deposition would be insignificant for C. japonica grown on dry sites. Unlike in C. japonica, water consumption in C. obtusa would be unlikely to respond to excess N deposition, regardless of the soil moisture level. Moreover, the significant reduction in the fine root to needle ratio observed with excessive N application in C. japonica under both Dry and Moist treatments suggests that excessive N deposition is likely to cancel out the tree's morphological adaptation to drought.