Inositol 1,4,5-triphosphate receptor protein immunohistochemistry of cerebellar Purkinje cells in two dogs with hypoglycemia

Immunohistochemical study was performed on cerebellar Purkinje cells of two dogs with hypoglycemia using an antibody against the inositol 1,4,5-triphosphate receptor that is identical to the cerebellar Purkinje cell glycoprotein P(400) (P(400)/InsP(3)R). In the cerebellar neocortex of an acute case of hypoglycemia, the P(400)/InsP(3)R staining of hypoglycemic Purkinje cells was heterogeneous: some peripheral dendrites, including spiny branchlets, were negative and others were stained with various intensities, although Purkinje cells were morphologically intact by hematoxylin and eosin (HE) stain. In a chronic case of hypoglycemia, almost all the dendrites of Purkinje cells of both the neo- and archicortex of the cerebellum were not stained with the P(400)/InsP(3)R antibody. This is in contrast to the normal dog where Purkinje cell bodies, axons, and dendrites, including spiny branchlets, are intensely stained by the P(400)/InsP(3)R antibody. These results suggest that P(400)/InsP(3)R immunolabeling of Purkinje cells decreased, despite their morphology being preserved by HE stain, and that the function of P(400)/InsP(3)R, especially in spiny branchlets that receive inputs originating from axon terminals of parallel fibers, may be impaired in hypoglycemia.     

Protectivity of an immunoaffinity-purified 39 kDa capsular protein of avian Pasteurella multocida in mice

A 39 kDa protein of avian Pasteurella multocida strain P-1059 (serovar A:3) was purified from a crude capsular extract by immunoaffinity chromatography by using a ligand of purified mouse monoclonal antibody to the 39 kDa capsular protein of the strain. Protective activity of the purified 39 kDa protein antigen was determined by inoculation of ddY mice twice with 25 or 125 mug of the protein and challenge-exposure with 10 or 50 LD(50) of strains P-1059 or X-73 (serovar A:1). The results showed that the antigen gave high protection (60 to 100%). These results indicated that the 39 kDa protein of avian P. multocida is a cross-protective antigen over serovars A:1 and A:3.     

Neospora caninum infected the alimentary tract of nude mice and was transmitted to other mice by intraperitoneal inoculation with the intestinal contents

Neospora caninum (BT-2 strain) that originated from the brain of a Holstein calf was serially passaged through 10 generations of BALB/c nude mice by intraperitoneal inoculation. Histological examination of the mice revealed that numerous clusters of tachyzoites appeared in the pancreas, stomach and small intestine as well as in the central nervous system (CNS) and skeletal muscles. Intestinal contents of the infected mice were inoculated intraperitoneally into uninfected nude mice and 3 of the 17 inoculated mice showed clinical signs at post inoculation days 3 to 10. The present experiments demonstrated a proliferation of N. caninum tachyzoites in the mucosa of the alimentary tract and pancreas of the nude mice and the intestinal contents of the mice were infective to other nude mice.     

Effects of recloning on the efficiency of production of alpha 1,3-galactosyltransferase knockout pigs

Obtaining sufficient transgenic cells via selective cultivation of genetically manipulated somatic cells is difficult due to the limited number of cell divisions. Additionally, if irreversible mutations in a cell's chromosomes occur during selective cultivation and the cell is used as the nuclear donor, somatic cell nuclear transfer (SCNT) embryos often exhibit abnormal development. On the other hand, a SCNT method in which fetal cells derived from SCNT embryos are used as the nuclear donor (recloning method) is an effective technique for obtaining large quantities of transgenic cells. In this study, we compared the in vivo development rate of SCNT embryos produced from porcine alpha1-3 galactosyltransferase gene knockout (GTKO) cells by a recloning method with that of SCNT embryos produced without recloning from porcine GTKO cells (direct method). In the direct method, 557 and 462 cloned embryos were produced using two types of activation methods, the two-step activation (TA) method and the delayed activation (DA) method, and then transferred into 6 and 4 recipients, respectively, but no piglets were born from these recipients. In the recloning method, 956 and 1038 cloned embryos were produced using the TA and DA methods, respectively, and then transferred to 8 and 7 recipients, respectively. Two piglets were born from one recipient in the TA group and 6 piglets were born from 3 recipients in the DA group. This report indicates that the recloning method improved the developmental capacity of SCNT embryos reconstructed with gene-targeted somatic cells.     

Airborne basidiospores as an inoculum source of <Emphasis Type="Italic">Typhula variabilis</Emphasis> and the effect of hilling on the incidence of Typhula winter rot of carrots

Typhula winter rot on overwintering carrots caused by Typhula variabilis is a newly confirmed disease, and no practical control measure is yet available. To develop a control method, here we researched the infection period of T. variabilis and the time that winter rot appeared on carrots. Using spore traps, we found that basidiospore rain occurred from September to November before snowfall in Memuro, Hokkaido. In addition, carrot leaves collected in autumn had already been infected by T. variabilis. These epidemiological investigations revealed that the pathogen releases basidiospores to infect carrot leaves before snow cover, resulting in root decay under snow. An effective control method was then developed to avoid direct contact of basidiospores of T. variabilis with plant tops by covering the plants with soil in autumn. Thus, the percentage of rotted roots was reduced to about half.     

Changes in extracellular neurotransmitters in the cerebrum of familial idiopathic epileptic shetland sheepdogs using an intracerebral microdialysis technique and immunohistochemical study for glutamate metabolism

Intracerebral microdialysis combined with electroencephalographic recordings was performed on 4 dogs of a familial idiopathic epileptic Shetland sheepdog colony to identify the kinds of neurotransmitters responsible for seizure activity. Immunohistochemistry using glutamate (Glu), glutamate transporter (GLT-1 and GLAST), and glutamine synthetase (GS) antibodies was also carried out on the cerebrum of four familial dogs that died of status epilepticus (SE). High values for extracellular levels of Glu and aspartate (ASP) were detected in association with an increased number of spikes and sharp waves during hyperventilation in 3 of 4 the familial epileptic dogs. The values of other amino acids analyzed were not altered in any of the familial epileptic dogs. Immunohistochemically, Glu-positive granules were occasionally found in the perineuronal spaces of the cerebral cortex in 3 of the familial epileptic dogs that died of SE. Immunostains for GLT-1 antibody predominantly decreased in the cerebral cortex and lateral nucleus of the thalamus in all the dogs that died of SE, whereas there were no differences detected in immunolabellings for GLAST and GS antibodies between familial epileptic dogs and controls. These results suggest that an extracellular release of both Glu and Asp may play an important role in the occurrence of seizure activity in this epileptic colony, and that a decreased expression of astrocytic GLT-1 may be related to development of SE.     

Occurrence of Pythium Ultimum Var. Ultimum in a Greenhouse on Spitsbergen Island, Svalbard

Pythium ultimum var. ultimum was isolated from carrot seedlings with damping off and from soil used for growing the plant in a greenhouse on Spitsbergen Island, Svalbard. The fungus caused severe damping off of carrot, cucumber and tomato seedlings after artificial inoculation. The rDNA internal transcribed spacer sequences of the Svalbard isolate were identical to those of Canadian and Japanese isolates of P. ultimum var. ultimum. The results suggest that the pathogen in the greenhouse on Svalbard was probably introduced from temperate regions through contaminated plants and/or soil imported to the island. This is the first record of P. ultimum var. ultimum within the Arctic zone.     

Model studies on precursor system generating blue pigment in onion and garlic

Reactions involved in blue-green discoloration in a mixture of onion (Allium cepa L.) and garlic (Allium sativum L.) were investigated. Vivid-blue color was successfully reproduced by using a defined model reaction system comprising only trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (1-PeCSO) from onion, S-allyl-L-cysteine sulfoxide (2-PeCSO) from garlic, purified alliinase (EC 4.4.1.4), and glycine (or some other amino acids). Four reaction steps identified and factors affecting the blue color formation were in good agreement with those suggested by earlier investigators. When crude onion alliinase was used in place of garlic alliinase, less pigment was formed. This result was explained by a difference in the amount of thiosulfinates, colorless intermediates termed color developers, yielded from 1-PeCSO by these enzymes.