INFORMATION: Theses for the Bachelor of Veterinary Medicine

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Title INFORMATION: Theses for the Bachelor of Veterinary Medicine

Citation Japanese Journal of Veterinary Research, 54(1), 47-79

Issue Date 2006-05-31

Doc URL http://hdl.handle.net/2115/13688

Type bulletin (article)

File Information 54(1)_p47-79.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

https://eprints.lib.hokudai.ac.jp/dspace/about.en.jsp

Characteristics of brown-like adipose tissue in mice with chronically elevated activity ofthe sympathetic nervous system.

Akihiro Uozumi

Laboratory of Biochemistry, Department of Biomedical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Mitochondrial uncoupling protein‐１（UCP１），a key molecule for metabolic thermo-genesis, is usually expressed only in brownadipose tissue（BAT）．White adipose tissue（WAT）converts into brown-like adipose tissueexpressing UCP１when animals are subjectedto chronic sympathetic nerve stimulation ,such as after cold acclimation. To clarify thethermogenic activity of brown-like adiposetissue, in this study, C５７BL６mice were in-jected with a highly selective β３‐adrenergicagonist, CL３１６，２４３（CL）or exposed to cold en-vironment at４℃．After２weeks under theseconditions, inguinal-WAT（I-WAT）turned intobrown-like adipose tissue expressing UCP１，but epididymal-WAT（E-WAT）did not. Adipo-cytes were isolated from I-WAT, E-WAT andBAT, and their oxygen consumption wasmeasured in vitro, as an index of thermoge-

netic activity. In adipocytes from brown-like I-WAT and BAT, but not those from E-WAT,oxygen consumption was increased in re-sponse to norepinephrine stimulation. Oxygenconsumption per UCP１ protein showed thatUCP１ in brown-like I-WAT had much higheractivity than that in BAT. Gene expression ofcell death-inducing DFF４５‐like effector-A（CIDEA），proposed as a UCP１inhibitor, wasalso increased in brown-like I-WAT . Theamount of UCP１ protein relative to CIDEAprotein（UCP１／CIDEA）was higher in BATthan in brown-like I-WAT, suggesting a minorrole of CIDEA in the control of UCP１activityin I-WAT.

Thus, brown-like I-WAT expressing UCP１，in addition to BAT, probably contributes towhole energy expenditure , because of itshighly thermogenic activity.

INFORMATION

Hokkaido University confered the degree of Bachelor of Veterinary Medicine to the following３８graduates of the School of Veterinary Medicine on March２４，２００６．

The authors summaries of their theses are as follows :

Jpn. J. Vet. Res. ５４（１）：４７‐７９，２００６

Proinsulin C-peptide modulates nitric oxide production and endothelial nitric oxidesynthase expression in the streptozotocin-induced diabetic rats.

Akihiro Kamikawa


C-peptide is cleaved off from proinsulinmolecule in the process of insulin biosynthesis,but stored in the secretory granule and re-leased into the blood circulation, along withinsulin. Recent studies have revealed that C-peptide is the biologically active peptideshowing ameliorative effects on diabetic com-plications such as neuropathy and nephropa-thy, possibly through nitric oxide（NO）produc-tion. Indeed, C-peptide stimulates NO produc-tion by activating enzyme activity and/or in-creasing enzyme amount of endothelial nitricoxide synthase（eNOS）in vitro . However, indiabetic patients and animal models in whichfunctions of endothelial cells are known to beimpaired by hyperglycemia , it is obscurewhether C-peptide induces NO productionand modifies eNOS. expression in vivo . Toclarify these issues, in the present study, I ex-amined plasma concentration of nitrogen ox-ides（NOx），metabolites of NO, and eNOS ex-pression in the kidney and lung in thestreptozotocin-induced type１diabetic modelrats, with or without replacement of either C-peptide or insulin by an osmotic pump. Ratstreated with streptozotocin showed hypergly-cemia with about ９０％ reduction of endoge-

nous insulin and C-peptide levels, but no ef-fect on plasma NOx concentrations, comparedwith normal rats. Diabetic rats treated withinsulin showed lower plasma glucose levelsthan that of diabetic rats treated with saline,but without affecting plasma NOx levels. Incontrast, diabetic rats treated with C-peptideincreased plasma NOx levels by３０％withoutinfluencing glucose levels . Diabetic ratstreated with saline dramatically increasedeNOS expression in the kidney, but not in thelung , compared with normal rats . Diabeticrats treated with either insulin or C-peptideabrogated the increase of eNOS expression inthe kidney and did not modify eNOS expres-sion in the lung . However , diabetic ratstreated with C-peptide by single bolus injec-tion failed to decrease the increase of eNOSexpression in the kidney. Therefore, the re-sults suggest that C-peptide is capable of in-ducing NO production in diabetic animals ,which may be attributed to amend abnormaleNOS expression in the kidney by improvingsystemic hemodynamics and reducing stressto kidney, while insulin suppresses the abnor-mal eNOS expression , possibly by reducinghyperglycemic stress to kidney.

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The relationship between UCP１（Uncoupling Protein１）and glucose uptake.

Chitoku Toda


Brown adipose tissue（BAT）is the majorsite of sympathetically mediated metabolicthermogenesis, at least in small rodents, dur-ing cold acclimation and spontaneous hy-perphagia . The principal substrate of BATthermogenesis is fatty acids, of which energyis dissipated as heat via uncoupling protein１（UCP１），a unique mitochondrial protein inBAT. It is known that glucose utilization inBAT is also increased in parallel with thermo-genesis. In the present study, to clarify thepossible relationship between UCP１ and tis-sue glucose utilization, I measured２‐deoxy-glucose（２DG）uptake into various tissues inmice acclimated to a cold environment（CA）or given a beta‐３ adrenergic agonist（CL３１６，２４３）daily（CL）for２weeks. Control miceexpressed UCP１only in BAT, whereas CA andCL mice expressed more UCP１in BAT and ec-topically in inguinal white adipose tissue（WAT），but little in epidydymal WAT. In con-

trol mice，２DG uptake in BAT was comparablewith those in heart under a basal conditionand increased after norepinephrine（NE）orinsulin stimulation. In contrast，２DG uptakein both types of WAT was undetectably lowand increased only by insulin stimulation. InCA and CL mice，２DG uptake under the basaland NE stimulation conditions increased inBAT, and remarkably in inguinal WAT, but lit-tle in epidydymal WAT. Although there weresome differences between CA and CL mice，２DG uptake in adipose tissues was roughlyparallel with the expression level of UCP１．Moreover, the stimulatory effect of insulin on２DG uptake was dramatically enhanced inWAT of CA and CL mice. All these results sug-gest that UCP１ activation may be beneficialfor the improvement of glucose metabolism,particularly under the condition of insulin re-sistance.

Changes in transient receptor potential V１function of dorsalroot ganglion neurons in mouse model of neuropathic pain

Ryuichi Komatsu

Laboratory of Pharmacology, Department of Biomedical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

I made the neuropathic pain model（Chronic Constriction Injury model : CCImodel） by sciatic nerve ligation （CCI-operation）using wild and transient receptorpotential V１knockout（TRPV１‐KO）mice, and

compared the paw withdrawal threshold tomechanical stimuli and paw withdrawal la-tency to thermal stimuli . To examine themechanism of hyperalgesia in vitro, I investi-gated changes in cytosolic Ca２＋concentration

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（［Ca２＋］i）in response to various stimuli usingfura‐２imaging system in dorsal root ganglion（DRG）neurons isolated from the CCI modelmice.

The paw withdrawal threshold to me-chanical stimuli was not different betweenwild and TRPV１‐KO mice before CCI-operation. CCI-operation decreased the pawwithdrawal threshold at the nerve-ligatedside（CCI-side），but not with the sham opera-tion side（sham-side）in both mice. At７‐dayafter CCI-operation, the decline of the pawwithdrawal threshold in TRPV１‐KO mice wassignificantly smaller than that in wild mice.The paw withdrawal latency to thermal stim-uli was significantly longer in TRPV１‐KOmice than wild mice before CCI-operation ,suggesting that thermal sensitivity is low inTRPV１‐KO mice. In both mice, CCI-operationshortened paw withdrawal latency, the extentof which was smaller in TRPV１‐KO mice thanwild mice . In DRG neurons isolated fromTRPV１‐KO mice, neither［Ca２＋］i response tocapsaicin nor TRPV１‐immunoreactive posi-tive cells were observed. RT-PCR revealed thelack of TRPV１mRNA in DRG of TRPV１‐KOmice . Capsaicin elicited significantly large［Ca２＋］i increase in DRG neurons at CCI-operated side（CCI-DRG）than those at sham-operated side（sham-DRG）．The percentageof cells with a large increment of［Ca２＋］i in re-

sponse to capsaicin was increased, but the to-tal number of cells responding to capsaicinwas unchanged by CCI-operation . In CCI-DRG, the increases of［Ca２＋］i in response tothermal stimuli and anandamide were signifi-cantly larger than in sham-DRG. On the otherhand , there was no difference in low pH-induced［Ca２＋］i increase between CCI-andsham-DRG .High K＋ or adenosine５’‐triphos-phate（ATP）induced［Ca２＋］i increase in CCI-and sham-DRG to the same extent .The in-crease of［Ca２＋］i induced by bradykinin（BK）was significantly larger in CCI-DRG thansham-DRG, and the percentage of cells with alarge increment of［Ca２＋］i in response to BKwas increased by CCI-operation．［Ca２＋］i re-sponses to BK were suppressed by a B２ an-tagonist but not a B１antagonist. A B１agonistfailed to evoke any［Ca２＋］i response in bothCCI-and sham-DRG . The potentiation of［Ca２＋］i response to capsaicin induced by BKwas significantly larger in CCI-DRG thansham-DRG. The number of IB４-negative cellswas increased by CCI-operation. In both CCI-and sham-DRG, there was no correlation be-tween IB４‐staining and responsibility to cap-saicin or BK.These results suggest that thepotentiation of TRPV１ function and changesin BK-induced regulation of TRPV１ throughB２receptor partly involve in neuropathic painphenotype in vivo.

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Characterization of voltage-dependent channels andinositol１，４，５‐trisphosphate-induced current

in porcine vomeronasal neurons

Ryo Takei

Laboratory of Pharmacology, Depatment of Biomedical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Using a whole-cell patch-clamp techniqueat a holding potential of‐９０mV, I examinedthe property of voltage-dependent channelsand the effect of putative second messengersof the pheromone in the vomeronasal neuronsof porcine vomeronasal slice preparations.

Voltage-dependent Na＋ channel currentswere transient inward currents which beganto be activated by depolarizing pulses to‐５０mV and reached a peak amplitude at‐３０mV.Tetrodotoxin（TTX）inhibited the currents ina dose-dependent manner. Voltage-dependentK＋ channel currents were outward and beganto be activated at depolarizing pulses to‐４０mV and were increased linearly with increas-ing depolarization. The currents were inhib-ited by tetraethylammonium（TEA）．Voltage-dependent Ca２＋ channel currents , mainlytransient inward currents, began to be acti-vated by depolarizing pulse to‐６０ mV andreached a peak at -４０mV. The currents wereinhibited by T-type Ca２＋ channel blockers, Ni＋

and mibefradil, but not inhibitited by an L-type Ca２＋ channel blocker, nifedipine . Sus-

tained inward currents were observed only１out of８cells in which transient Ca２＋currentsoccured.

Extracellular application of arachidonicacid or a diacylglycerol analog, and intracellu-lar application of Ca２＋ or cAMP using cagedcompounds did not induce any currents. In８out of１３ cells dialyzed with caged inositol１，４，５-trisphosphate（IP３），IP３released by pho-tolysis evoked inward currents. The latency ofthe current responses was２．０±０．７ sec, andthe average amplitude of a maximal currentwas -７２．８±２２．４pA. A reversal potential of IP３-induced currents was -２９．２±３．５mV. The IP３-induced current was not observed in cellsdialyzed with Cs＋.

These results indicate that porcine vo-meronasal neurons have several voltage-dependent channels such as TTX-sensitiveNa＋ channels, TEA-sensitive K＋ channels andT-type Ca２＋ channels. Furthermore, it is sug-gested that intracellular IP３ activates chan-nels in porcine vomeronasal neurons.

Jpn. J. Vet. Res. ５４（１），２００６ Information ５１

Hypercapnic acidosis-induced adenosine release from spinal cord of neonatal rat

Masaaki Ban

Laboratory of Pharmacology, Depatment of Biomedical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

In order to reveal the mechanisms ofadenosine release induced by hypercapnic aci-dosis, we examined the effect of artificial cere-bral spinal fluid gassed with２０％ CO２（hy-percapnic acidosis ACSF）on adenosine re-lease from the isolated spinal cord of neonatalrats . We also investigated the changes ofadenosine kinase activity during hypercapnicacidosis. The pH of the ACSF gassed with５％CO２＋９５％ O２（normal ACSF）and hypercap-nic acidosis ACSF was７．３ and６．７，respec-tively. The hypercapnic acidosis increasedadenosine release from the spinal cord but notaffected on adenine nucleotide release . Al-though hypoxia（５％ CO２＋９５％ N２）increasedadenosine release, the ACSF gassed with５％CO２＋１５％ N２＋８０％ O２had no effect on adeno-sine release.

An ecto‐５’‐nucleototidase inhibitor, ARL６７１５６，had no effect on hypercapnic acidosis-induced increase in adenosine release. On theother hand, homocysteine, which trapped in-tracellular adenosine, depressed the increasein adenosine release caused by hypercapnicacidosis. Tetrodotoxin and removal of extra-cellular Ca２＋ had no effect on the increase ofadenosine release by hypercapnic acidosis. Anequilibrative nucleoside transporter（ENT）in-hibitor, NBTI, had no effect on basal adeno-

sine release , and inhibited the increase ofadenosine release by hypercapnic acidosis .Another ENT inhibitor , dipyridamole , in-creased basal adenosine release and de-pressed the increase in adenosine release byhypercapnic acidosis.

Hyperphoshate acidosis ACSF（５％ CO２，pH ６．７） and isohydric hypercapnia ACSF（２０％ CO２，pH７．３）increased adenosine re-lease from the spinal cord. However hypocap-nic acidosis ACSF（５％ CO２，pH６．７）and acidi-fied HEPES ACSF（pH６．７）had no effect onit. An adenosine kinase inhibitor, ABT７０２，in-creased adenosine release from the spinalcord. Hypercapnic acidosis or hyperphosphateacidosis on failed to cause further increases inadenosine release in the presence of ABT７０２．Hypercapnic acidosis（pH６．７），hyperphos-phate acidosis（pH６．７）and isohydric hyper-capnia（pH７．３）depressed adenosine kinaseactivity in rat spinal cords.

These results indicate that hypercapnicacidosis causes the accumulation of intracel-lular adenosine and then adenosine releasevia NBTI-sensitive ENT, resulting from theinhibits adenosine kinase activity in neonatalrat spinal cords. The inhibition of adenosinekinase activity may be induced by a fall of in-tracellular pH.

Jpn. J. Vet. Res. ５４（１），２００６Information５２

Vaccine efficacy of the common antigens of Leptospira interrogans

Takayuki Kamikawa

Laboratory of Microbiology, Department of Disease Control,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Leptospirosis is a zoonotic infectioncaused by pathogenic Leptospira interrogansand occurs worldwide. Vaccines with inacti-vated whole cells are used in the prevention ofthis disease, but these vaccines induce a se-rovar specific immunity and are not effectivefor infections with different serovars. Thus,outer membrane proteins LipL４１，LipL４５andLipL３２that are the common antigens of L. in-terrogans were expressed and purified as re-combinant proteins to examine the effects ofserovar-independent vaccines.

Genes of LipL４１，LipL４５and LipL３２werecloned and inserted into expression plasmids.Recombinant proteins were expressed in a fu-sion form with a Histidine tag in Escherichiacoli . The recombinant proteins were purifiedwith histidine-affinity columns . Four-week-old C３H/HeJ mice were subcutaneously im-munized , two times , at two-week intervalsand were subsequently challenged intraperi-toneally with L. interrogans serovar Manilaestrain UP-MMC for examination of vaccine ef-

ficacy.Expression and purity of the recombinant

LipL４１，LipL４５ and LipL３２ proteins wereanalyzed as anticipated molecular mass. Se-rum antibody titers of almost all the micewere elevated by immunization with the re-combinant protein. In the groups immunizedwith single recombinant proteins, some micelived１or２days longer than the negative con-trols after challenge. Furthermore, three micethat were immunized with a mixture of threerecombinant proteins lived１ day longer orsurvived for１４days. Moreover, the fact thatno leptospira was isolated from the kidneys ofmice was an indication of the efficacy of thevaccines.

These results suggest that recombinantproteins when used in combination resultedin enhanced vaccine efficacy. Further researchis required to determine a new antigen that ismore effective and for the development of amethod of immunization for the improvementof vaccine efficacy.

Antigenic and genetic analyses of H５influenza virus isolates from aquatic birds for vaccineand diagnostic use against highly pathogenic avian influenza

Kosuke Soda

Laboratory of Microbiology, Department of Disease Control,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Outbreaks of highly pathogenic avian in-fluenza（HPAI）caused by H５N１viruses haveoccurred in fourteen Asian countries includ-

ing Japan since２００３and caused serious eco-nomic loss. The aim of this study was to pro-duce an effective vaccine prepared with a vi-

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rus strain antigenically and genetically simi-lar to the currently circulating HPAI virusesin Asia for the occurrence of outbreaks thatwould not be able to control by test andslaughter in future in Japan. In order to es-tablish a vaccine strain, genes and antigenic-ity of H５influenza viruses isolated from feralducks and swans, the natural reservoir of in-fluenza A virus, were analyzed. Effects of thetest vaccines were evaluated by the animalexperiment with chickens.

Twelve H５ influenza viruses were iso-lated from the fecal samples of aquatic birdsin Hokkaido and Mongolia during１９９６‐２００４，and their hemagglutinins（HAs）were ana-lyzed antigenically and genetically deter-mined. Phylogenetic analysis of H５HA genesrevealed that eleven isolates were classifiedinto Eurasian type and the other was classi-fied into North American type and that cleav-age site of all isolates were LP profile. Homol-ogy of amino acid sequences of the HAs be-tween isolates and HPAI viruses was over９０％ . A panel of monoclonal antibodies

against H５HA was used for the antigenicanalysis of isolates and HPAI viruses. It wasindicated that a close similarity in the anti-genicity between HPAI viruses and isolates ofaquatic birds-origin. Inactivated test vaccineswith oil adjuvant were prepared from A/R /Hokkaido／１／０４（H５N１），whose HA gene wasderived from A/duck/Mongolia／５４／０１（H５N２），one of the parent viruses used for the produc-tion of the reassortant. The other parent vi-ruses were A/duck/Mongolia／４７／０１（H７N１）and A/duck/Hokkaido／４９／９８（H９N２）. Effectsof the test vaccines were investigated by chal-lenge experiment against chickens. Antibodytiters of immunized chickens were dependenton the concentration of antigen in the testvaccines. The chickens which had over４HIantibody titers by vaccination survived chal-lenge with HPAI virus, A/chicken/Yamaguchi／７／０４（H５N１）．

From these results, low pathogenic H５in-fluenza viruses isolated from aquatic birdswere useful as development of vaccines.

Characterization of male factor derived from Rhipicephalus appendiculatus

Yuko Ito

Laboratory of Infectious Diseases, Department of Disease Control,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Tick feeding activities and transmissionof pathogens cause great losses in the live-stock industry. Since ticks play importantroles as vectors of various pathogens, suppres-sion of their population is the most effectiveway to control the disease which they trans-mit, particularly protozoan diseases. At pre-sent, ticks can be effectively controlled by theuse of acaricides, which have many disadvan-tages. Hence, it is necessary to develop alter-

native tick-control methods such as immu-nological way, which is currently consideredas a major sustainable and practical method.

Previously, it was reported that Voraxinderived from Amblyomma hebraeum wastransferred from male to female ticks duringcopulation as a male factor（MF），and stimu-lated the females to engorge. In addition, rab-bits immunized with the recombinant Voraxinshowed anti-tick immunity. Thus, the objec-

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tive of this study was to obtain Voraxin-likeMFs from Rhipicephalus appendiculatus ,which is a vector for East coast fever.

Total of２８７ cDNAs were cloned from acDNA library constructed from fed male ticks.One cDNA, named as RAMF１，has a sequencesimilarity to the Is5 gene expressed in maleIxodes scapularis, but not in female. RT-PCRanalysis showed that RAMF１was expressedspecifically in the gonads of fed males. Micro-injection of the recombinant RAMF１（rRAMF１）into the hemocoel of virgin female

ticks resulted in prolonged feeding time andslight increase in body engorgement of theseticks compared to those of control virgin fe-males . Vaccination of guinea pigs withrRAMF１did not show any effects on ticks ex-cept that the average of the egg weight per en-gorged body weight was smaller than that ofthe control ticks. These results suggested thatRAMF１，which is not a Voraxin-like molecule,might be one of the factors that support fe-males to engorge.

Epidemiological investigation of Newcastle disease virus and avian influenza virus isolatedfrom fecal samples of wild ducks and geese

Takashi Sato


Newcastle disease virus（NDV）and avianinfluenza virus（AIV）have been responsiblefor serious economic losses in the poultry in-dustry. These viruses were isolated from vari-ous free-living-birds（ducks, pigeons, parrotsetc）．Therefore, wild birds are considered asreservoirs and vectors of NDV and AIV. Thiswork aims to investigate the distribution ofthese viruses in wild ducks at Hokkaido.

In this study, from total２，９２７ fecal andcloacal swab samples of wild ducks and geesecollected in Hokkaido in２００３and２００４，６NDVstrains，８AIV strains（１of H３N２，６of H３N８，１of H４N６serotypes），５avian paramyxovirus４（APMV４）strains and２APMV６strains wereisolated. These AIVs and APMVs, except for

NDV, are usually isolated from wild ducks,and are non-pathogenic to chickens. Althoughall６isolated NDV strains showed low patho-genicity to chicken embryos，２out of６isolatedNDV strains contained a pair of dibasic aminoacids at the cleavage site of the fusion protein,which is a characteristics of a virulent type ofNDV, suggesting that they are potentiallypathogenic. The result of phylogenetic analy-sis indicated that these NDV isolates belongto Genotypes�and�，suggesting lentogenicand old types of NDVs are maintained in wildducks. Further investigation is expected fordetailed understanding of these viruses inwild birds.

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Application of simple genotyping method for bovine viral diarrhea virus, analysis oftissue distribution of the virus in the in the infected cattle and seroepidemiology

of free-living deer in Japan

Yumiko Nishikura


Bovine viral diarrhea virus（BVDV）infec-tion induces various clinical symptoms in theinfected cattle including diarrhea, abortion ,and respiratory and neurological symptoms.BVDV infection during the first semester ofgestation period may result in the birth ofpersistently infected（PI）cattle. Four geno-types of BVDV，１a，１b，１c and２，are popular inJapan, and BVDV lc can be isolated from cat-tle showing neurological deterioration. In thepresent study, application of a simple geno-typing for BVDV was performed using polym-erase chain reaction-restriction fragmentlength polymorphism（PCR-RFLP）instead ofa general nucleotide sequence analysis.

Using the combination of restriction en-zymes, EcoO１０９I and NsiI, BVDV lc was dis-tinguished from other genotypes. However, afew BVDV la viruses, which were mutated atthe restriction enzyme sites, were classifiedinto BVDV lc by PCR-RFLP. This simple geno-typing method could be useful for easy and

rapid screening of BVDV lc although there isdisadvantage of misclassification of someBVDV la strains.

Tissue distribution of BVDV lc in PI cat-tle was also analyzed to know the relation ofBVDV lc with the occurrence of neurologicaldisorder. Results of virus quantification of re-vealed that there was no difference betweenBVDV lc and other genotypes, and that allBVDV genotypes were present in all thetested organs . Interestingly, immunohisto-chemical staining showed the presence of theviral antigen in neurocytes of cerebrum of twoPI cattle which were infected with BVDV lc.

Since BVDV lc was isolated from free-living deer, serologica１survey of BVDV in Ja-pan was conducted by using １４７ Sika deer（Cervus nippon）samples．BVDV was not iso-lated from those samples, but２of those sam-ples had low titers of neutralizing antibodyagainst BVDV la.

Diagnosis of Echinococcus multilocularis infection in definitive hostsby copro-DNA detection

Sota Kamihiro

Laboratory of Parasitology, Department of Disease ControlSchool of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan

Echinococcus multilocularis is an impor-tant zoonotic parasite which causes alveolar

echinococcosis in humans. The increasing riskof the infection to companion animals as well

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as humans is anticipated in Hokkaido, Japan,because the prevalence of the parasite in wildfoxes has increased dramatically in the lasttwo decades. To evaluate the risk of E. multi-locularis infection and to take effective pre-ventive measures, it is necessary to accuratelydiagnose the infection in the definitive hosts,especially in companion animals which are incloser contact with humans. At present, stan-dard diagnostic procedure for the definitivehosts is detection of antigen and eggs in theirfeces , followed by detection of the parasiteDNA from the eggs. However, no eggs can bedetected while the coproantigen is positive, incase of prepatent or light infection. Positiveresults of the coproantigen test alone cannotbe conclusive evidence because of its cross re-activity to other taeniid parasites and occa-sional false positive result. Hence, to confirmthe diagnosis , it is essential to detect theparasite DNA from the feces even when theeggs cannot be detected.

In Chapter � , a method to detect theparasite DNA from eggs was evaluated usingthree canine feces, which were positive forcoproantigen and taeniid eggs in the screen-ing test for E. multilocularis infection. TheirEPG（eggs per gram）were１，４and８０，respec-tively. The eggs were separated from one gramof the each feces by floating and sieving tech-nique. DNA was extracted from the eggs us-ing QIAamp DNA Mini Kit and amplified by aspecies-specific single tube nested PCR for E.multilocularis. The target sequence for ampli-fication was a part of the E. multilocularis mi-tochondrial １２S ribosomal RNA gene . Allthree samples were positive for the PCR andtheir infections were confirmed. This method

was sensitive enough to detect the parasiteDNA from the sample which contained onlyone egg per gram.

In Chapter �, detection of DNA from fe-ces of infected animals which contained noeggs, especially feces during the prepatent pe-riod of the infection were evaluated . Ninedogs and two cats were orally given１，０００ to１，０００，０００protoscoleces and the feces of theseanimals were collected daily. DNA was ex-tracted from the feces using QIAamp DNAStool Mini Kit and amplified by the nestedPCR. At０‐２days after infection, feces of fivedogs and two cats were positive for the PCR.The detected DNA was probably derived fromthe unestablished protoscoleces or debris ofthe cysts in the inoculated materials. How-ever, DNA was detected only sporadically af-ter the early phase of the infection, indicatingthat DNA examination with this technique isnot reliable enough to detect the infectionduring the prepatent period.

Finally, in Chapter �, detection of DNAfrom feces during the prepatent period incombination with praziquantel treatment wasexamined. It was expected that feces after thetreatment would contain more tissues of theparasite than that of non-treated dogs. Threedogs were orally given１０，０００or１００，０００pro-toscoleces and were treated with praziquantelat１２or１４days after infection. Soon after thetreatment, the DNA was detected from the fe-ces of all three dogs.

These results suggested that the DNA ex-amination from feces in combination with an-thelminthic treatment could become a newoption for the diagnosis of E. multilocularisinfection in definitive hosts.

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Genetic analysis of heat shock resistance in AKR/N mouse spermatogenesis

Daisuke Torigoe

Laboratory of Experimental Animal Sciences, Department of Disease Control,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Generally in mammalian testis , a tem-perature lower than the abdominal tempera-ture by２‐８℃ is required for the normalspermatogenesis . It is known that in cryp-torchid testis, germinal cell loss caused by theheat-stress results in male sterility. However,the mechanism of germinal cell loss in cryp-torchidism is not well understood. Recently, ithas been found that strain difference is pre-sent in the response to heat stress in mousetestis（Kazusa et al．，２００４）．The MRL/MpJmouse has an abnormal exonuclease 1（Exo1）gene, which was suggested to be the cause ofheat-stress-resistant phenotype of the testis（Namiki et al．，２００５）．The AKR mouse, whichis one of the originated strains of the MRL/Mpmouse, also shows heat-stress-resistant phe-notype. Thus, we attempted to identify geneticloci responsible for the heat-stress-resistantphenotype in the AKR/N mouse by quantita-tive trait loci（QTL）analysis.

Testis weight ratio at３weeks after ex-perimental cryptorchidism in C５７BL／６J micewas significantly smaller than AKR/N mice.We investigated the expression of Exo1 genein AKR/N mice to check whether the heat-stress-resistant phenotype in the AKR / Nmouse was caused by mutation of the Exo1gene. However AKR/N mice showed normalExo1 gene expression. Testis weight ratio in

（C５７BL／６J×AKR/N）F１mice was also signifi-cantly smaller than that of AKR/N mice. Incontrast, there was a remarkable variation intestis weight ratio in F２mice, suggesting thatheat-stress-resistant phenotype is controlledby multiple genetic loci . A genome-wideanalysis of linkage with testis weight ratio re-vealed two weak QTL on chromosome（Chr）１０near D10Mit16（１６cM）and D10Mit42（４４cM）loci. We could not detect the significantQTL on Chr１near the Exo1 gene in consis-tent with the result that the expression of Exo1 gene was normal in the AKR/N mouse testis.There are some genes involved in apoptosisand spermatogenesis , such as Tbpl1, Apaf1and Perp on Chr１０．Some of these genes mayhave mutations or polymorphisms as causingthe heat-stress-resistant phenotype in theAKR/N mouse. We could not detect other QTLexcept for those in Chr１０．These results indi-cate that multiple genes, including those onChr１０and those on the other chromosomes,cooperatively contribute to the phenotype ofthe AKR/N mouse.

We expect that further investigationwould contribute to the understanding of themechanism of spermatogenesis and to the de-velopment of the therapy for the infertilitycaused by heat stress in both human and ani-mals.

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Epidemiological survey and pedigree analysis of GM１gangliosidosis in Shiba dogs andneuronal ceroid lipofuscinosis in Border collies using rapid mutation screening.

Natsuko Kawahara

Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

In hereditary diseases in animals, if themutation responsible for a disease is identi-fied and a rapid mutation screening methodfor the disease is developed, the genotypes ofindividuals can be determined, and heterozy-gous carriers can be found. Inherited diseasescan be prevented and controlled by eliminat-ing heterozygous carriers used for breedingpurposes. In this study, as part of the preven-tive program for GM１gangliosidosis in Shibadogs and neuronal ceroid lipofuscinosis（NCL）in Border collies, we simplified the mu-tation screening methods for both diseases,and an epidemiological survey and pedigreeanalysis were carried out using these meth-ods.

In mutation screening（PCR-RFLP : po-lymerase chain reaction-restriction fragmentlength polymorphism）for GM１gangliosidosisin Shiba dogs, the conditions of the methodusing buccal swab specimens were examined.PCR products from specimens are sufficientfor genotyping when using a specialized com-mercial kit for collecting buccal mucosal cells,even if buccal mucosa are swabbed only once,and if the swabs are stored at room tempera-ture for５days after swabbing. We collectedbuccal swab specimens from ８１ Shiba dogs

that belong to the Shiba Dog Club in theCzech Republic . As a result of genotypingthese dogs, all the genotypes of the dogs werenormal. This method was considered suitablefor mass analysis of the genotypes.

For mutation screening for NCL in Bor-der collies , we established PCR-RFLP andPCR-PIRA （ primer-introduced restrictionanalysis）methods. The PCR-RFLP methodwas useful for determining NCL-affected dogs,but not for distinguishing normal dogs fromheterozygous carriers , in contrast with thePCR-PIRA method . We therefore concludedthat it was necessary to use these methods to-gether for genotype analysis.

Genotyping of８of the１１ Border colliesdiagnosed with NCL in Japan was performedusing the２methods described above. As a re-sult, all the dogs were shown to have the mu-tation homozygously, which has been reportedin other countries as a causative mutation ofNCL. Moreover, as a result of analyzing pedi-gree information, it was shown that most ofthese dogs were blood relatives. The results ofthis study suggested that the NCL mutationhas been distributed in Japan due to the in-breeding of imported dogs.

Jpn. J. Vet. Res. ５４（１），２００６ Information ５９

Induction of phase II detoxification enzymes by alk（en）ylthiosulfates derived from Allium plants

Miyan Ko

Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Two alk（en）yl thiosulfates, sodium n-propyl thiosulfate（NPTS） and sodium 2-propenyl thiosulfate（２PTS），are natural con-stituents of onion and garlic, respectively, andwere identified originally as causative agentsof onion-and garlic-induced hemolytic anemiain dogs. Recently, it has been demonstratedthat onion and garlic have an inhibitory effecton carcinogenesis in experimental animalsdue to the increased tissue activities of phaseII detoxification enzymes. In this study, wemeasured the activities and mRNA expres-sion of phase II detoxification enzymes in cul-tured H４IIE rat hepatoma cells which weretreated with several concentrations of NPTSand ２PTS. The effects of diallyl trisulfide（DATS）and tert-buthlhydroquinone（TBHQ），known as phase II detoxifying inducers, onthe enzymes were also examined and com-pared with those of alk（en）yl thiosulfates.Furthermore, NPTS and２PTS were examinedfor their ability to prevent X-ray-induced

DNA damage using the comet assay.As a result，２PTS was found to signifi-

cantly increase quinone reductase（QR）activ-ity, and QR and epoxide hydrolase１（EH１）mRNA expressions compared with the control.However, glutahione S -transferase（GST）ac-tivity, and GSTA５ and UDP-glucuronosyltransferase１A１（UDPGT１A１）mRNA expres-sions were not changed by２PTS. In contrast,NPTS did not induce any phase II enzymes.DATS and TBHQ significantly increased themRNA of QR, GSTA５and EH１．２PTS has anequal or higher potential for inducing phase IIenzymes compared to DATS and TBHQ. Inaddition, NPTS and２PTS protected against X-ray-induced DNA damage in H４IIE.

From these results, it was demonstratedthat２PTS could induce phase II enzymes, andthat NPTS and２PTS had a radioprotective ef-fect, suggesting that alk（en）yl thiosulfateshave an effect in preventing cancer.

Effects of ionophore compounds, valinomycin and nystatin,on intracellular Babesia gibsoni viability in vitro.

Norihisa Tamura

Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.

The effects of two ionophore compounds,valinomycin and nystatin, on babesia parasite（Babesia gibsoni）viability were investigated

in vitro.B. gibsoni was cultured with normal dog

erythrocytes containing high Na＋ and low K＋

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concentrations and lacking Na, K-ATPase（LKdog erythrocytes）．When valinomycin andnystatin were added to the culture , respec-tively, the number of intracellular parasiteswas decreased, indicating that each ionophorehad an anti-babesial effect on B. gibsoni in vi-tro. On the other hand, the concentration ofK＋ and Na＋ in LK dog erythrocytes was un-changed by the ionophores. These results sug-gested that valinomycin and nystatin mightdirectly injure B . gibsoni within LK dogerythrocytes.

To clarify the mechanism of ionophoresagainst the parasite, the effects of valinomy-cin and nystatin on HK dog erythrocytes wereexamined. HK dog erythrocytes have high K＋

and low Na＋ concentrations through the func-tion of Na, K-ATPase, which presents geneti-cally in their membranes . When HK dogerythrocytes were incubated with the iono-phores, the concentration of K＋ was decreasedand that of Na＋ was increased with a decreaseof intracellular adenosine ５’‐triphopahate（ATP）concentration. Furthermore, HK dogerythrocytes were hemolyzed during incuba-tion. When Na, K-ATPase was inhibited withouabain, however, the concentration of intra-cellular ATP did not change and no hemolysisof HK dog erythrocytes was observed duringthe incubation of HK dog erythrocytes with

each ionophore. These results suggested thateach ionophore might activate an ion pump,such as Na, K-ATPase on the cell membrane,resulting in the depletion of ATP in the cells.ATP depletion seemed to affect cellular viabil-ity.

Finally, to clarify whether the cytosol of B.gibsoni contained high K＋ and low Na＋ con-centrations, the anti-babesial effect of eachionophore against B. gibsoni parasite withinLK dog erythrocytes was examined using aculture medium consisting of high K＋ and lowNa＋ concentrations . As a results , no anti-babesial effect of each ionophore was observedin that medium. This result might suggestthat the ion pump in the cytoplasm of B. gib-soni might not be activated in medium con-taining high K＋ concentration, and that thecytosol of B. gibsoni might contain high K＋

and low Na＋ concentrations, indicating thatthe parasite might have its own sodium pumpto maintain high K＋ and low Na＋ concentra-tions.

In this study, it was suggested thatvalinomycin and nystatin might activate theion pump of B. gibsoni through perturbationof the cation gradient in the parasite cytosol,resulting in ATP depletion of the parasite .This may be a mechanism of the ionophoresagainst B. gibsoni.

Functional assessment of equine nectin‐１as an entry receptor for equine herpesvirus１

Shin’ichi Igawa

Laboratory of Comparative Pathology, Department of Veterinary Clinical SciencesSchool of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Equine herpesvirus１（EHV‐１）is knownas a causative agent of equine rhinopneu-monitis , abortion , and encephalomyelitis .

EHV‐１has caused serious problems in horseindustries worldwide. The effect of inactivatedvaccine currently used in Japan is short-

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termed. In addition, the vaccine is not able toinduce a cellular immune response that isstrong and effective. To conquer EHV‐１infec-tion , more effective prophylaxes and treat-ments have been desired. Therefore, it is nec-essary to identify viral and host factors in-volved in the development of the EHV‐１infec-tion.

Although the receptor for EHV‐１has notbeen identified, five receptor molecules thatmediate the entry of other alphaherpesvi-ruses have been well characterized so far.Among those receptors, nectin‐１act as a com-mon receptor for Herpes simplex virus‐１，Her-pes simplex virus‐２，Pseudorabies virus, andBovine herpesvirus‐１．It has been reportedthat the human nectin‐１molecule does notmediate entry of EHV‐１ into cells. However,the function of equine nectin‐１as an receptorfor EHV‐１ remains to be elucidated. In this

study, a cDNA of equine nectin‐１（eNec‐１）open reading frame was cloned from equinebrain microvascular endothelial cells. The de-duced amino acid sequence of eNec‐１showedhigh degree of similarities（ greater than９０％）with that of human, porcine, bovine andmurine nectin‐１．The eNec‐１had two uniqueamino acid substitutions（P５６L and I８０L）within a putataive virus-binding region of theV domain.

In order to assess a function of eNec‐１asan entry receptor for EHV-１，we transfectedeNec‐１into NIH３T３cells that were unsuscep-tible to EHV‐１infection, and then transfectedcells were exposed to the recombinant EHV‐１expressing GFP. This virus did not infect theNIH３T３ cells expressing eNec‐１．These re-sults suggest that EHV-１，unlike other alpha-herpesviruses, is not able to use nectin‐１ asan entry receptor.

Age-dependent resistance of developing rat to Japanese encephalitis virus infection

Taro Nagashima

Laboratory of Comparative Pathology, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Fischer rats infected intracerebrally withJapanese encephalitis virus（JEV）show age-specific mortality ; rats infected at１２days ofage or younger result in１００％ mortality andrats infected at１４days of age or older resultin１００％ survival. This age-related resistancehas been shown to be related to neuronal ma-turity, since developing rat neurons, but notmature ones, were demonstrated to be the tar-get of JEV. However, the events underlyingthis dramatic change in susceptibility havebeen poorly understood.

Fischer rats were infected with JEV Ja-GAr‐０１strain intracerebrally at the age of４，

８，１０，１２and１７days. Histologically, distribu-tions of necrotic foci and JEV antigen-positivecells were almost the same and were more re-stricted in older rats. Brain virus load corre-lated inversely with increasing age at thetime of inoculation. Clearance of infectious vi-rus from the CNS delayed in younger animals.Although all rats except the ones inoculatedat４days of age eventually developed serumantibody response after infection , compari-sons showed no significant differences in anti-body titer among rats inoculated at８，１０，１２and１７days of age. In some rats infected at８or１０days of age resulted in moderate to se-

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vere thymic atrophy , indicating systemicstress response. These data suggest that themortality of rats from JEV infection is related

to brain virus load, and also suggest the possi-bility of involvement of systemic stress re-sponse in the mortality.

Experimental demonstration of transneural spread of Listeria monocytogenes

Tomohisa Tanaka

Laboratory of Comparative Pathology, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Listeria（L.）monocytogenes causes liste-riosis in humans and domestic animals. Insheep and other ruminants which have devel-oped encephalitis after L. monocytogenes in-fection, the lesions are usually confined to thebrain stem, especially the pons and medullaoblongata . One of the explanations for thepredilection to the brain stem is that L. mono-cytogenes spreads directly from peripheralsites to the brain stem via cranial nerves.

In this study, L. monocytogenes was in-oculated to right cheek muscles of mice andthe brains, cranial nerves and visceral organsof the mice were examined histologically andimmunohistochemically. To evaluate the tran-saxonal spread of L. monocytogenes in vitro,nerve cells from murine dorsal root gangliawere cultured in compartmentalized culturesystem, inoculated with the bacteria to axons,and then investigated using confocal laser mi-croscope whether the bacteria were able to as-cend the axons. Erysipelothrix（E.）rhusiopa-

thiae was used as negative control.Inoculation of L . monocytogenes to

murine right buccinator induced ipsilateralrhombencephalitis and neuritis of thetrigeminal and facial nerves. Bacterial anti-gen was detected in those lesions by immuno-histochemistry. In the compartmentalized cul-ture system, total number of L. monocytogenesmoved from axon terminal to nerve cell bodyincreased with time and many of them ap-peared to be located within axons. E. rhu-siopathiae also moved to the compartment ofnerve cell body along axons, but many of themappeared attaching to the axonal surface un-der the laser microscope.

In conclusion, it was suggested that L.monocytogenes was able to spread to centralnervous system via peripheral nerves such astrigeminal and facial nerves, and the transax-onal passway might be one of the route oftransneural spread of L. monocytogenes.

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Simplified diagnostic procedures for an alteration of endometrial epidermal growth factorprofile during the estrous cycle in repeat breeder cows

Noboru Takaesu

Laboratory of Theriogenology, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Repeat breeder cows that fail to conceiveafter three or more inseminations without ap-parent abnormalities in their genital tractsand estrous cycles have been a major source ofeconomic loss in dairy herds. Changes in en-dometrial epidermal growth factor（EGF）con-centrations are altered in some of repeatbreeder cows. Although normal cows have twopeaks of endometrial EGF concentrations inthe estrous cycle, some repeat breeder cows donot have them. Detection of the alteration hasbeen used to segregate this type of repeatbreeders from those with unknown causes ofinfertility to choose the type of treatment .However , the current diagnostic protocolneeds repeated biopsy of endometrial tissuesand EGF assay requires tissue processingwhich is time consuming and labor intensive.Thus, the present study evaluated, first, thepotential of vaginal curage materials as alter-nate materials to estimate endometrial EGFconcentrations and, then, use of Sep-Pak C１８cartridge to simplify the EGF separation pro-

tocol.EGF concentrations of vaginal curage

materials per total protein showed a similarchange to endometrial EGF concentrationsthat had２peaks on Days２to４and１４of es-trous cycle and EGF concentrations betweenthe２materials highly correlated（y＝１．２１x－０．７３，R＝０．９８３２，P＜０．０５）．Separation of EGFin endometrial tissues using Sep-Pak C１８ car-tridge（Sep-Pak method）was equally effectiveto the conventional method using SephadexG-５０ column when５０ or１００％ methanol or８０％ acetonitrile were used for elution. Therewas a positive correlation between EGF con-centrations of vaginal curage and endometrialmaterials separated by the Sep-Pak methodwhen５０％ methanol was used for elution（y＝０．９０x＋３．４７，R＝０．６１５４，P＜０．０５）．Theseresults indicate that endometrial EGF profilecan be estimated by using vaginal curage ma-terial and the EGF separation protocol withSep-Pak C１８with５０％methanol for elution.

In vitro ovulation of in vivo grown follicles isolated from eCG-treated mice :effects of hCG dose in culture medium and initial follicle size.

Ryohei Nakamura

Laboratory of Theriogenology, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

The objective of this study was to exam-ine the effects of human chorionic gonadtro-

phin（hCG）dose supplemented with culturemedia and initial size of follicles on efficiency

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of ovulation in vitro. In vivo grown follicles（３５０‐６５０µm of diameter）were isolated fromthe ovaries of mice treated with equine chori-onic gonadtrophin（eCG）. Some of the folli-cles were recovered after４８ h of eCG treat-ment（eCG group），and the others were recov-ered３h after hCG injection given４８h aftereCG treatment（eCG/hCG group）．The iso-lated follicles were cultured for２１h（eCG/hCGgroup）or２４h（eCG group）in media contain-ing５，１０or２０IU/ml of hCG to examine theirovulation and nuclear status of ovulated andnon-ovulated oocytes. The concentrations ofsex steroid hormones in the culture media foreCG group were also measured.

In the eCG/hCG group, ovulation rate ofthe follicles cultured in a medium containing５IU/ml of hCG was lower than that of１０and２０ IU / ml . Follicles with lager initial sizeshowed lower ovulation rate , and follicleswith more than６００ µm of diameter did notovulate. In the eCG group, no difference wasobserved in ovulation rate between the folli-cles cultured with１０and２０IU/ml of hCG, butfollicles with more than５００ µm of diameter

did not ovulate. There was no difference in theovulation rate between eCG / hCG and eCGgroups when the follicles with３５０‐５００µm ofinitial diameter were subjected to culture .Most ovulated oocytes were at the metaphse�in the eCG/hCG（７４％）and eCG（９４％）groups. Some（３０％）of the oocytes of non-ovulated follicles in eCG/hCG group, but noneof the oocytes in eCG group, showed the re-sumption of meiosis. Estradiol concentrationsof the follicle culture media of ovulated folli-cles was higher than those of non-ovulatedfollicles. Eighty percent of ovulated folliclesproduced high level of estradiol or estradiol/progesterone, and７０％ of non-ovulated folli-cles produced high levels of progesterone ortestosterone , suggesting that non-ovulatedfollicles were in the process of atresia . Thepresent results indicate that supplementationof follicle culture media with１０IU/ml of hCGis necessary to induce in vitro ovulation of thefollicles recovered from eCG / hCG-treatedmice. A low ovulation rate in the follicles withlarger initial sizes indicates the necessity ofmodification in the follicle culture conditions.

A novel posttranslational modificationwith４-hydroxyl-２-nonenal of the major red cell membrane skeletal protein β-spectrin

Mira Yang

Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Red cell membrane proteins are known tobe susceptible to form the adducts with a lipidperoxidative product ４-hydroxyl-２-nonenal（４-HNE）．However, it remains to be clarifiedwhich proteins are the targets of this modifi-cation and how it occurs. The present studyreports a novel finding that spectrin, the ma-jor constituent of the red cell membrane

skeleton, is the primary protein accessible to４-HNE. When human red cell membrane pro-teins and crude spectrin preparations wereanalyzed for the ４-HNE adducts by im-munoblotting, profound signals were observedin α-and β-spectrin and were remarkably in-creased after exposure of the membranes to４-HNE. No significant differences were ob-

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served for the signals of４-HNE adducts ofspectrin polypeptides in immunoblots of mem-branes from red cells separated on anarabinogalactan-density gradient centrifuga-tion . The MALDI-TOF mass spectrometryanalysis for tryptic peptides derived from in-tact β-spectrin demonstrated that４-HNE wasadded to some peptides including Ile１１０－Lys１１８，Val２９３－Lys３０２，Ala４８０－Lys４９７，and Leu１２３４－Lys１２４４

localized in the N-terminal actin/protein４．１‐binding domain（βN），the segment１，segment２，and segment１０，respectively, of β-spectrin.The modification of Ile１１０－Lys１１８ with４-HNEwas also found in the４５-kDa polypeptide pro-duced by limited digestion of β-spectrin withV８ protease followed by mass spectrometricanalysis. Glutathione S-transferase-fused pr-

oteins of the βN, segments２-４（β［２-４］），andsegments５-７（β［５-７］）of β-spectrin were gen-erated and were analyzed by mass spectrome-try. Surprisingly, the４-HNE adducts of thepeptides Ile１１０－Lys１１８ and Ala４８０－Lys４９７ werefound in the βN and β［２-４］，respectively, whileno adducts were detected in β［５-７］，suggest-ing that the modification also occurred in bac-terial cells. Considering the previously sup-posed interactions of the βN and β［２-４］re-gions with phosphatidylserine, these findingssuggest that the posttranslational modifica-tion of β-spectrin is a spontaneous event sub-sequent to polypeptide synthesis, presumablyby the association of the protein with mem-brane lipids, rather than a change during thered cell senescence.

Molecular mechanisms for down-regulation ofthe α-hemoglobin stabilizing protein（AHSP）gene expression in prion diseases

Kayoko Katsuoka


α-Hemoglobin stabilizing protein（AHSP）is an erythroid-specific molecular chaperonethat stabilizes newly synthesized α-globin .The previous study demonstrated that mRNAlevels of AHSP was specifically reduced in he-matopoietic tissues of PrPSc-infected animals.The purpose of the present study was to clar-ify the mechanisms for down-regulation of theAHSP gene expression in erythroid precursorcells. The MELhide８ clone, exhibiting highlevels of hemoglobin production in response toerythroid differentiation induced with N,N’-diacetyl-１，６-diaminohexane（HMBA），was es-tablished from the parental MEL cells andwas found to possess PrPC and generate AHSPwhen induced by HMBA. The effects of PrPSc

and several inflammatory cytokines on theexpression of some erythroid-specific genesincluding AHSP were examined in this cloneand the cells over-expressing PrPC（MELhide８-MoPrP）．The addition of brain homogenatesfrom scrapie-infected mice to HMBA-inducedMELhide８ cells had no effect on the expres-sion of AHSP, α- , and β-globins , GATA-１，EKLF, NF-E２，and band３．No accumulation ofPrPSc in MELhide８-MoPrP cells was foundeven after１６passages, while N２a cells over-expressing PrPC showed profound accumula-tion of PrPSc. Interleukin（IL）‐１β，Tumor ne-crosis factor（TNF）-α，and IL‐６principallycaused suppression of AHSP gene expressionat the concentrations reported for sera in hu-

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mans and animals suffered from prion dis-eases, while these cytokines had no effect onthe expression of the band３gene. However,the decrease in the AHSP gene expression ap-peared not enough to cause the reduction inAHSP production, since no significant changewas found in AHSP contents in erythroid cells

from the animals inoculated with PrPSc. Thesefindings suggest that some inflammatory cy-tokines, but not the PrPSc , can cause down-regulation in expression of the genes charac-teristic to erythroid cells including AHSP inprion diseases.

Physiological roles of band３（anion exchanger１）in assembly of the red cell membrane skeleton :a study for production of transgenic mice with band３expression at an early stage of

erythroid development

Toshihiro Takeda


Little is known about the molecularmechanisms for the assembly of red cell mem-brane skeleton during the development andmaturation of erythroid cells. Previous stud-ies have shown that membrane skeletal pro-teins are stably assembled into the membranein late erythroblasts concomitant to the onsetof band３，anion exchanger１，expression. Thepurpose of the present study was to establishthe transgenic mice with enforced expressionof band３in early stages of erythroid differen-tiation under the regulation of a novelerythroid-specific promoter of GATA-１，a tran-scription factor characteristic of erythroid li-nage. Unfortunately, various murine band３mutants, containing the HA- or FLAG-tag se-quence in the３rd or４th extracellular loop,failed to target the plasma membrane whentransfected into HEK２９３or MEL cells. Thus,the cDNA for the wild-type mouse band３orthe mouse-bovine chimeric band３was in-serted downstream the GATA-１ promoterIE３．９int and was transferred into embryos.Subsequent analyses for１７ individual mice

produced showed that the transgenes were in-corporated in their genomic sequences. Quan-titative PCR analyses for bone marrow cellsfrom F１animals showed that the two out of１７parental transgenic mice had increased ex-pression, by６to１２ times, of the transgenescompared with the endogenous band３geneexpression. It was also demonstrated that, un-der transgenic situation , both lin－TER１１９－

and lin－TER１１９＋ erythroid cells exhibited ex-pression of the band３gene, consistent withthe expression pattern of GATA-１，whereasband３ expression was predominant in thelin－TER１１９＋ population and negligible inlin－TER１１９－ cells in normal mice, suggestingthat the IE３．９int promoter displayed itsproper function. However, no red cell pheno-types and/or no significant expression of theband３protein derived from the transgenewere observed in these individuals, suggest-ing that the promoter function was notenough to induce the gene expression suffi-cient to cover large amount of band３．

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Changes in the expression of claudins along with the developmental stagesin the murine mammary epithelia

Keitaro Morishita


Claudins are the major constituents ofthe tight junction in the epithelial cells. Asthe first step to establish the molecular basesfor different susceptibilities to mastitis indairy cows, the changes in the expression ofvarious claudins in distinct stages of mam-mary glands were investigated in mice by RT-PCR, quantitative PCR, immunofluorescencemicroscopy , and immunoblotting . RT-PCRanalysis demonstrated that claudins １，３-１２，１５，１７，and １９ were expressed in mousemammary glands with relatively high levelscompared with other claudins. QuantitativePCR exhibited that claudin-８and -１２had re-markable increase in the transcripts in thelactating period, in which the levels of geneexpression of claudin-１，-４，and -１０ were re-duced. Claudin-３and -５appeared to be consti-tutive, since their expressions were kept at

constant levels despite of significant altera-tion in tissue architecture in different stages.Compatible changes in relative abundance ofthese claudin proteins were also demon-strated in immunoblotting, i.e., claudin-３and-８were predominant in the lactating period,while the content of claudin-８ was remark-ably reduced in non-lactating stages. More-over, immunofluorescence microscopy showedthat claudins３，４，５，８，and１０were exclusivelydistributed to the tight junction , whileclaudin-１was localized to the basement mem-brane . These findings demonstrate thatclaudin-３and -８are the major components oftheight junctions in murine mammary glandsand suggest that the developmental changesof several claudins including claudin-８ playkey role in regulation of permeability barrierfunction in the mammary gland.

Development of RT-PCR to detect tick-borne encephalitis virus genomic RNA

Yumiko Saga

Laboratory of Public Health, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

In October １９９３，a human case of en-cephalitis was diagnosed as tick-borne en-cephalitis（TBE）in Kamiiso, Hokkaido. Far-Eastern subtype TBE virus strain Oshima５‐１０ was isolated from a sentinel dog in thesame area. Since the suspected vector ticksand reservoir rodents are commonly found in

Japan, TBE virus may be endemic not only inthe area where a patient was found but alsoin other parts of Japan. Seroepidemiologicalsurvey for wild rodents is efficient to detectTBE endemic area . Now, virus-neutralizingtest（NT）is used for serological survey of wildrodents. But neutralizing antibodies can’t be

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detected at initial phase of TBE infection .TBE infection at initial phase can be diag-nosed by detection of virus RNA. In this study,I developed reverse transcription-polymerasechain reaction（RT-PCR）to detect TBE virusRNA.

There are three main subtypes of TBE vi-rus ; the European, Far Eastern, and Siberiansubtypes. Therefore I designed the PCR prim-ers to detect all subtypes of TBE virus. Thisprimers set could detect each subtype of TBEvirus, specifically. The detection limit of thisRT-PCR was at least１０２FFU/tube. In addition,it was examined whether this method coulddetect virus RNA in animal tissues. RT-PCRfor tissues from experimentally infected mice

could detect viral RNA in the blood, spleen,and brain samples.

Next, this RT-PCR was applied to the di-agnosis of TBE virus from wild rodents. Thespleen and serum samples were collected inNakagawa , Hokkaido , which is a potentialTBE endemic area. There is no positive sam-ple in RT-PCR , but６ samples had TBE-specific antibodies, indicating that Nakagawawas endemic for TBE virus.

In summary, I developed RT-PCR thatcan detect TBE viral RNA from tissue of ro-dent. Combine with serological study, this RT-PCR can be useful to specify TBE endemicarea.

Characterization of monoclonal antibodies to nucleocapsid protein ofsevere acute respiratory syndrome（SARS）coronavirus

Hiroshi Noda


Severe acute respiratory syndrome（SARS）suddenly emerged from the end of２００２，originated from Guangdong Province inChina, and expanded to the large part of theworld. The epidemic ended in July２００３withtotal patient number more than８，０００and７７４deaths. The causative agent was quickly iden-tified as the distinct coronavirus called SARS-coronavirus（SARS-CoV）．Since the reservoiranimal has not been determined and no effec-tive vaccine and antiviral agent have been de-veloped, the most effective measure to pre-vent the expansion of SARS-epidemic may berapid diagnosis and isolation of SARS pa-tients. Therefore, establishment of specific di-agnostic methods is crucially important . Toestablish a reliable diagnostic method，９

clones of monoclonal antibodies（MAb）to nu-cleocapsid protein（NP）of SARS-CoV weregenerated.

All９clones of MAb were produced by im-munization of recombinant NP or a syntheticpeptide of SARS-CoV NP（SARS-NP）to mice.All MAb reacted with a Western blot analysis（WB）． In addition , no MAb had cross-reactivity to authentic NP of human coronavi-rus（HCoV）２２９E strain or recombinant NPof HCoV２２９E strain by WB.

To determine the binding region of MAbon NP , four truncated recombinant NP（trNP）were generated and reactivity betweenMAb and trNPs were analyzed by WB andELISA. It was revealed that２MAb recognizedtrNP２region（１１１‐２３０aa）and７MAb recog-

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nized trNP３region（２２１‐３４０aa）．The epitopes of MAb on NP were deter-

mined by Fmoc method, in which variety ofamino acid sequences of NP were synthesizedon a membrane. It was revealed that MAb SN５‐２５ recognized amino acid sequence“QTVTKK”on SARS-NP（２４５‐２５０aa）and rSN１２２recognize“LPYGAN”（１２２‐１２７aa）．These

amino acid sequences of the epitopes weresearched in the database and no such se-quences were found within proteins of maincausative agents of human respiratory illness.Therefore, it is strongly suggested that these２MAb are specific to SARS-CoV and usefulfor diagnostic reagents.

Development of new diagnostic methods of hantavirus and its epidemiological application.

Yoichi Tanikawa


Hantaviruses are causative agents of se-vere zoonotic diseases which are hemorrhagicfever with renal syndrome（HFRS）and han-tavirus pulmonary syndrome（HPS）．Varioustypes of hantaviruses are distributed all overthe world in their specific natural rodenthosts.

In Hokkaido and Far East Russia, grayred-backed voles〈Clethrionomys(C. )rufoca-nus〉have hantavirus that is related to Puu-mala type hantavirus. In this study, the de-tailed genetic analysis of Sakhaline strain ofhantavirus which was isolated from C. rufoca-nus captured in Sakhaline were performed.

Comparison of nucleotide sequences andphylogenetic analysis among hantaviruses re-vealed that Sakhaline strain was most relatedto Puumala type hantavirus but may be clas-sified in a different type within the genusHantavirus.

Antigen detection ELISA and quantita-tive real-time PCR were developed for new di-

agnostic methods of hantavirus infection .Hantavirus replication was analyzed inPuumala-infected Syrian hamsters（Mesocricetus auratus）．Hamsters were in-fected with Puumala virus at４week old asadult. The virus antigen and RNA peaked at１４days post infection（d.p.i）and the virusRNA was detected even at７０d.p.i. The modeof infection in Syrian hamster was similar tothat of the natural hosts. Therefore, Puumala-infected Syrian hamster is a suitable modelfor hantavirus infection in vivo.

In addition, antigen detection ELISA andReal-time PCR were applied４８C. rufocanuscaptured in Nakagawa town, Hokkaido to un-derstand the mode of virus infection in natu-ral hosts. Virus RNA was detected from５se-ropositive animals. In addition, virus antigenand RNA were detected from２seronegativeanimals. Therefore, these methods are suit-able to analyze the mode of hantavirus infec-tion in natural host in detail.

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Antioxidant activity of mouse prion protein

Tetsu Inoue

Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

The cellular prion protein（PrPC）is a gly-cosylphosphatidylinositol（GPI）-plasma mem-brane-anchored protein. Since octarepeat do-main（OR domain）in the amino-terminal re-gion of PrPC acts as a Cu２＋-binding domain,Cu２＋ ion is considered to modulate variousbiological functions of PrPC, i.e., the cellularenzymatic activity of superoxide dismutase（SOD），signal transduction, shedding of PrPC

and conversion to the scrapie isoform of prionprotein（PrPSc）．In this study, to clarify furtherphysiological function of PrPC as an antioxi-dant, the susceptibility of PrPC-overexpressedcells to various oxidants was evaluated. In ad-dition , we prepared recombinat mouse PrPand examined its SOD-like activity and theinhibiting activity against Cu２＋ / H２O２ -catalyzed oxidative reaction or Cu２＋-catalyzedoxidative reaction of tBuOOH in cell-free sys-tem.

Wild-type moPrP（moPrP（２３‐２３１））was over-expressed in mouse embryo fibroblastNIH３T３ cells using pRc / EF-MoPrP vectorwith Lipofectamine and the cell viability aftertreatment of various oxidants was assessed byWST‐１assay. In the assay for cell-free anti-oxidant activity of PrP, recombinant mousePrP（ rmoPrP（２３‐２３１））and truncated PrP（rmoPrP（９１‐２３１）），lacking OR domain, were ex-pressed using E. coli expression system, andthe folded rmoPrP was purified by Ni２＋-resin

and reverse-phase HPLC. The O２－-scavengingactivity（SOD-like activity）of rmoPrP and theinhibiting activity of rmoPrP against Cu２＋/H２

O２ -catalyzed oxidative reaction or Cu２＋-catalyzed oxidative reaction of tBuOOH weremeasured by spin-trapping technique usingelectron spin resonance spectroscopy（ESR）with spin trapping agent，５，５-dimethyl‐１‐pyrroline-N-oxide（DMPO）．

In NIH３T３cells overexpressed with wild-type PrP, the cell viability after treatment ofoverload of Cu２＋，H２O２ and ionizing radiationwas measured . The Cu２＋ -and H２O２-inducedcell death of NIH３T３ cells were significantlyinhibited by overexpression of PrPC , butradiation-induced cell death was not. On theother hand, in the assay for cell-free antioxi-dant activity of PrP, ESR-spin-trapping ex-periments showed that the SOD-like activityof rmoPrP（２３‐２３１）/Cu２＋ complex was about twotimes higher than that of Cu２＋ alone, but１０３

times lower than that of Cu/Zn-SOD. The oxi-dative reaction of Cu２＋ with H２O２ or Cu２＋ -catalyzed oxidative reaction of tBuOOH wasalso shown to be significantly reduced by theaddition of rmoPrP（２３‐２３１） but not rmoPrP（９１‐２３１）by spin-trapping experiments. These resultssuggested that PrPC inhibited the cellulardamage induced by Cu２＋ -and H２O２-oxidativestress thorough OR domain of PrP.

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Visualization of formalin- and capsaicin-induced pain and assessment ofanalgesics in rat cerebral cortex.

－Application of high magnetic field BOLD-fMRI－

Taketo Uemura


Blood oxygen level dependent-functionalMRI（BOLD-fMRI）is imaging technique em-ploying the difference in the oxygenation levelof hemogrobin which is an endogenetic con-trast agent. It is useful as non-invasive way tovisualize locally activated area in brain aftervarious stimulations. In this study, the possi-bility that the fMRI method can visualize aresponse of pain was examined in rat cerebralcortex . In order to demonstrate that BOLDsignal changes actually reflect the pain in thecerebral cortex , we compared BOLD-signalswith morphine with those without morphine.Experiment was performed using a７．０５Teslasuperconducting MRI system, Varian UnityINOVA３００／１８３，and a one-turned surface coilcentered over the primary somatosensory cor-tex in cerebral cortex of rat under the me-chanical ventilation. A set of multi-slice gradi-ent echo images was acquired and analyzedusing commercially available software for im-aging analysis（MEDx）． In contrast to α-chloralose anesthetization , which is widelyused as an anesthetic in forepaw stimulationin fMRI study of rats, isoflurane（１．０％）pro-vided the stable anesthesia level and the fa-vorable results concerning the fMRI in ratcerebral cortex . Following the subcutaneousinjection of５０µl of formalin（５％）or capsicin（１０µg/head）into the left forpaw, a regional in-crease in the signal intensity in the MR im-ages was observed in all rats.

Formalin stimulation to the left forepaw

of rat displayed that the signal increase（about６％）was observed in contralateral so-matosensory cortex following its injection, itwas maintained for４minutes. Furthermore,the continuous but weak signal increase（about２％）in signal was observed for２０min-utes after stimulation. Pre-treatment（３mg/kgi.v.）with morphine vanished these responses.Capsaicin stimulation in left forepaw dis-played that the signal increase（about５％）was observed immediately after stimulation,but the signal disappeared within one minute.Pre-treatment（３mg/kg i.v.）with morphinevanished these responses in a similar fashionto the formalin stimulation.

It is well known that BOLD signal de-pends on the cerebral blood flow. In eithercase of formalin or capsaicin, temporary riseof systemic blood pressure was observed, butthis change of blood pressure was not com-pletely depressed in administration of mor-phine , whereas BOLD signal was not ob-served in rats administered with morphine.The rise of systemic blood pressure might notnecessarily affect the changes in BOLD signalof brain. Accordingly, it was suggest that thechanges in BOLD signals in somatosensorycortex certainly reflected the change of focalblood flow. This BOLD-fMRI technique inanesthetized animal brain is a useful way tostudy the mechanism of pain and assess newanalgesics.

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Effects of combined treatment of novel anti-tumor nucleosides with X irradiationon the apoptosis induction in MKN４５cells

Yu Sato


Recently, two anti-tumor drugs，１‐（３‐C-ethynyl-β-D-ribo-pentofuranosyl）cytosine（ECyd）and１‐（２‐deoxy‐２‐methylene-β-D-erythro-pentofuranosyl）cytosine（DMDC），were newly developed to induce apoptotic celldeath by inhibiting RNA synthesis and DNAsynthesis , respectively. Since several anti-tumor drugs such as cisplatin and５‐FU wereknown to have radio-sensitization effects inclinical radiotherapy, we examined whetherECyd and DMDC have radio sensitization ef-fects on the apoptosis induction in humangastric adenocarcinoma MKN４５ cells. Obser-vation of morphological changes in nuclei re-vealed that treatment with ECyd alone in-duced the dose-dependent increase of apopto-sis in MKN４５ cells , although X irradiationalone did not. Moreover, combined treatmentof ECyd with X irradiation increased apopto-

sis in comparison with treatment with ECydalone. On the other hand, DMDC had no radio-sensitization effects in the apoptosis induc-tion. Flowcytometric and western blotting ex-periments showed that ECyd abrogatedradiation-induced G２／M checkpoint and accu-mulation of phosphorylated Cdc２，whereasDMDC did not affect these radiation-inducedphenomena. Furthermore, ECyd was shownto reduce the expression of Bcl‐２but not Bcl-XL in MKN４５cells with X irradiation, whereasDMDC did not change the expression of theseanti-apoptotic proteins. These results suggestthat ECyd as an RNA synthesis inhibitor ispreferable to DMDC as a DNA synthesis in-hibitor when treatment of anti-tumor drug iscombined with X irradiation for inducingapoptotic cell death in therapy for solid tumor.

Novel Mechanism of Warfarin Resistance in Roof Rats（Rattus rattus）of Tokyo

Fumie Okajima

Laboratory of Toxicology, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Roof rats are often found in high-risebuildings in urban areas. Attempts to exter-minate them with rodenticides are failing andtheir numbers are increasing in the Tokyoarea . An increase in the population ofwarfarin-resistant rats is proposed to be thereason. Recent investigations report the in-

volvement of vitamin K epoxide reductasecomplex subunit１（VKORC１）gene mutationin this resistance.

Using liver microsomes of warfarin-resistant（R）rats from Shinjuku and warfarin-sensitive（S）rats from a closed colony（origi-nally from the Ogasawara Islands） for the

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control, I measured vitamin K epoxide reduc-tase（VKOR）and vitamin K quinone reduc-tase（VKR）activities and their sensitivitiesto warfarin in both groups of rats. I found nosignificant difference between them in VKRactivity. Resistant rats showed significantlylower VKOR activity than sensitive rats .VKOR activity in sensitive rats was markedlydecreased with warfarin, while only a slightdecrease in the already-low activity was ob-served in resistant rats. I found that purifiedNADPH cytochrome P４５０reductase（fp２）hasVKOR activity. Fp２activity determined by cy-tochrome c reductase activity in microsomesof resistant rats was３‐８ times higher thanthat in sensitive rats. These results suggestthat fp２functions as a VKOR enzyme in resis-tant rats to compensate for reduced VKORC１activity.

The warfarin level in serum（AUC）was

significantly lower in warfarin-resistant ratsafter oral administration than in the controlwarfarin-sensitive rats . Concentrations ofwarfarin metabolites in urine were higher inwarfarin-resistant rats than those in warfarin-sensitive rats. Hydroxylations of warfarin bycytochromes P４５０（CYP）isoforms were signifi-cantly higher in warfarin-resistant rats. West-ern blot analysis indicated that levels of CYP３A２expression in warfarin-resistant rats weresignificantly higher than in warfarin-sensitive rats.

It is concluded that the mechanism ofwarfarin resistance in Tokyo roof rats in-volves high fp２-dependent VKOR activitycompensating for low VKOR enzyme activityand increased clearance of warfarin due to in-creased microsomal activities of cytochrome P４５０reductase and CYP３A２．

Synergistic toxicity between dopamine-derived neurotoxin, norsalsolinol, and transitionmetals, Implication for etiology of Parkinson’s disease

Yoshihiro Ozaki

Laboratory of Toxicology, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Parkinson’s disease（PD） is one of theneurodegenerative diseases that causes motordisorders. Most of PD is reported to be spo-radic. There are three major hypotheses of eti-ology : the dopamine quinone theory, the inhi-bition of mitochondrial complex I theory, andthe oxidative stress theory. Since Langston etal. reported that１‐methyl‐４‐phenyl‐１，２，３，６‐tetrahydropyridine（MPTP）causes Parkin-sonism in１９８３，many studies were carried outto find the causative substance, but it remainsunknown . Recently, tetrahydroisoquinolines（ TIQs），whose structures are similar to

MPTP, are suspected as possible causativefactors for PD.

Norsalsolinol is one of the TIQs and canbe biosynthesized from dopamine and formal-dehyde by Pictet-Spengler condensation inthe brain. In this study, I investigated the celltoxicity of norsalsolinol and its capability ofcausing PD with in vivo and in vitro experi-ments . According to many reports , metalssuch as iron or copper are accumulated in thebrains of PD patients. Thus, I also investi-gated the toxicity of these metals with andwithout norsalsolinol.

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Rat midbrain primary cultures and PC１２cells, which were induced to neuron-like cellsby NGF, were exposed to these chemicals and/or metals. A marked decrease of cell viabilityand increased production of ROS were ob-served. In particular, norsalsolinol and metalsshowed a synergistic effect in primary cells,producing ROS up to２．７-fold and reducingcell viability to about５０％ compared to１００％in controls.

In vivo studies revealed that spontaneousmotor activities were significantly decreasedin continuously norsalsolinol ＋ iron-infused

rats. Significant decreases of dopamine con-centration in the midbrain（６８±１０％）were ob-served in these rats. Those changes were notfound in iron-infused rats.

A previous report showed that norsalsoli-nol is actively transported by the dopaminetransporter, implying that norsalsolinol pro-duces toxicity in the substantia nigra. Takentogether, these results suggest that a syner-gism between norsalsolinol and metals inneurotoxicity may contribute to the onset ofPD.

Pharmacokinetics of Glivec（imatinib）in dogs

Sumiko Nagai

Laboratory of Toxicology, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sappro 060-0818, Japan

Glivec�（imatinib mesylate）is a ration-ally designed, potent and highly selective ty-rosine kinase inhibitor. It is marketed for thetreatments of chronic myeloid leukaemia（CML）and gastrointestinal stromal tumours（GISTs）in human. Mast cell tumors（MCT）are the most common neoplasm in the skin ofdogs, and half of these tumors are malignant.Glivec has the possibility to inhibit the func-tion of abnormally activated tyrosine kinasein MCT cells of dogs. However, the pharma-cokinetics of imatinib in dogs have not beenreported. The aim of this study is to deter-mine in vitro and in vivo pharmacokinetics ofimatinib in dogs.

In Chapter１，I established the method ofdetermination of Glivec using HPLC（high-pressure liquid chromatography） equippedwith a UV detecter.

In Chapter ２，the pharmacokinetics ofGlivec in plasma of dogs were investigated,

using the method in Chapter１．Glivec wasorally or intravenously administered to eightdogs . The time of the peak concentration（Tmax）of imatinib was４to９hours, indicat-ing the similarity to Tmax in humans. I foundlarge individual differences in the maximumconcentration of imatinib in plasma（Cmax,C０），the area under the plasma concentrationversus time curves（AUC）and elimination half-life（t１／２）．After oral administration, Cmaxranged from０．５２to３．０µM（n＝６）and aver-age value（±SD）was１．４±１．０µM, AUC（±SD）was９５０±７２０µM・min , t１／２（±SD）was１３５±１２３min. After intravernous injection , C０ranged from８．１to３４．４µM（n＝８）and aver-age value（±SD）was１５．５±８．３µM, AUC（±SD）was９９０±４００µM・min, t１／２（±SD）was１９６±１２０min .The pharmacokinetic parame-ters in dogs exhibited higher similarity withthose in the monkeys than parameters in hu-mans. Binding affinities of plasma proteins to

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imatinib are lower in dogs and monkeys thanthat in humans. Moreover, I found that thebioavailability of Glivec in dogs was low（７１±５１％）compared with that in humans（９８％）．

In Chapter３，cytochrome P４５０（CYP）mo-lecular species which metabolize Glivec indogs were identified. The metabolic activitiesin the reconstruction system of dog CYP３A１２and CYP２C２１，and inhibition of CYP３A de-pendent activity using anti-rat CYP３A anti-body and ketoconazol, showed that CYP３A１２mainly metabolized Glivec in the liver micro-some of the dog.

It is reported that CYP３A subfamily con-tributes to the metabolism of numerous clini-cal medicines in dogs. Vitamin D receives me-tabolism by CYP３A in human, and it is clini-cally used for transitional cell carcinoma and

osteosarcoma of dogs. Then, in Chapter４，Istudied the possibility of adverse drug inter-action between Vitamin D and Glivec. In vivo ,I did not find any differences in plasma phar-macokinetics between Glivec and Glivec-vitamin D treated dogs. I also showed thatthere is no effect of vitamin D on Glivec me-tabolism in dog liver microsomes in vitro.

In present study, I report pharmacokinet-ics of Glivec in dogs, and the P４５０molecule（CYP３A１２）which mainly contributes to themetabolism of Glivec. In addition, I found thatvitamin D does not interfere with pharma-cokinetics and metabolism of Glivec. These re-sults may contribute to the establishment ofeffective therapeutic use of Glivec in veteri-nary field.

Growth-related changes in gonadal histology and immunohistochemistry of theimmature green turtle（Chelonia mydas）

Saori Otsuka

Laboratory of Wildlife Biology, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

This study aimed to reveal growth-related histological changes of green turtle（Chelonia mydas）gonads and to establish away to identify the growth stages of the seaturtles. The role of steroid hormones in thegrowth of sea turtle gonads was also dis-cussed from an immunohistochemical study ofsteroidogenic enzymes（P４５０scc，３βHSD, P４５０c１７，and P４５０arom）and steroid hormone re-ceptors（AR, ERα, ERβ, and PR）．

Testes can be categorized into six stagesby histology, and here there was a wide rangeof straight carapace lengths（SCL）for eachstage. However, there were no large changesin efferent ductules and epididymal ducts .

Steroidogenic enzymes were immunolocalizedin Leydig cells, and different kinds of steroidhormone receptors were expressed within thesame cells in both testes and epididymides,while immunoreaction of all receptors was en-hanced as the stages advance.

Ovaries and oviducts were distinguish-able between immature turtles and those inpuberty, as was immunolocalization of steroidhormone receptors within them . Differentsteroid hormone receptors were expressed inthe same cells as observed in male gonads.

These results suggested that size is not areliable indicator of maturity ; however, itwas possible to histologically identify the

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growth stages in males. Although it was notpossible to categorize the growth stages of im-mature females, there was an indicated thatmaturation of ovaries and oviducts are syn-chronized and that a histological search is notessential to distinguish between immaturity

and puberty. Since the gonads of both sexesbecame sensitive to these steroid hormonesduring growth, it was suggested that interac-tions between these hormones regulate gonadmaturation and function.

Prevalence of antibodies to the hepatitis E virus（HEV）in pigs and cattle in Hokkaido

Yuko Miya

Laboratory of Wildlife Biology, Department of Environmental Veterinary Sciences,School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan

Recently, hepatitis E has drawn attentionas a zoonosis, and there have been severalcases reported in Japan that suggest food-borne transmission from pigs, deer, and boar.These animals are thought likely to be reser-voirs of HEV. Some cases suggesting HEV in-fection from pigs to humans have been re-ported in Hokkaido ; thus we tested pigs andcattle in Hokkaido for the presence of anti-HEV IgG and analyzed the associated publichealth risks. Some previous studies have beenundertaken utilizing ELISA, but without theuse of proper control serum. In this study, ex-perimentally immunized sera from pig anddeer were prepared and used as the respectivepositive control sera for ELISA, and Westernblot assay was used as the secondary test. Theresults showed a prevalence of３．６％ in pigs ;suggesting that the actual prevalence and

risks of food-borne transmission were not asgreat as previously reported. Nevertheless, in-vasion of HEV was confirmed and thus it isnecessary to pay attention that raw meat isnot consumed. In cattle, there was no anti-HEV IgG among those tested ; suggestingthat cattle present little risk of HEV infection.However, if cattle were to be infected withHEV, it would be transmitted to deer as theyshare the same pastureland and so would be-come widespread. HEV infection in cattle de-serves further attention, and periodic inspec-tion of cattle together with surveillance ofdeer is required. As ELISA is likely to includesome false positive reactions, we propose thata combination of ELISA screening with West-ern blot assay confirmation is a good tech-nique for large-scale serological surveillanceof HEV.

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Frequencies of PrP genotypes in Japanese sheep flocks－for breeding programs for TSE resistance in Japanese sheep－

Jiro Ohara

Laboratory of Prion Diseases, School of Veterinary Medicine,Hokkaido University, Sapporo 060-0818, Japan

There are many amino acid polymor-phisms in ovine PrP. Two of them, polymor-phisms at codons１３６（A/V）and１７１（Q/R）greatly influence the susceptibility to scrapie.Sheep possessing AQ/VQ or AQ/AQ genotypeare susceptibile to scrapie, while sheep pos-sessing AQ/AR genotype are relatively resist-ant and those possessing AR/AR are highlyresistant to scrapie. To reveal the susceptibil-ity of Japanese Suffolk sheep to scrapie, inthis study, the author analyzed the frequen-cies of PrP genotypes in Suffolk sheep. In ad-dition, to understand the efficiency of estab-lishing scrapie-resistant sheep flocks by se-lecting the sire based on their PrP genotypes,the author analyzed the transition of the fre-quencies of PrP genotypes before and afterthe implementation of the selective breedingat Shintoku Hokkaido Animal Research Cen-ter（HARC）．To investigate the amino acidpolymorphisms in PrP of Suffolk sheep, DNAfragments corresponding to the open readingframe of the PrP gene were amplified fromgenomic DNA isolated from ９８ sheep andtheir nucleotide sequences were determinedby direct sequencing. Nucleotide substitutionsresulting in amino acid substitution werefound at codons１１２（M/T），１３６（A/V）and１７１（Q/R）．Thus, in the following analyses, the

author carried out a single nucleotide poly-morphisms analysis for codons１３６ and１７１that are related to scrapie-susceptibility. Thefrequencies of PrP genotypes varied amongregions in Japan ; the frequencies of sheeppossessed scrapie-susceptible genotypes AQ/VQ or AQ / AQ were ranging from ４２．９ to６３．１％．Moreover, genotype frequencies for AQ/ VQ or AQ / AQ varied from ３０．９ to １００％among the private sheep flocks in Hokkaido.These results suggested that the ratio ofscrapie-susceptible Suffolk sheep differedamong regions and flocks. A year after initiat-ing the selective breeding using rams carry-ing scrapie-resistant AQ/AR or AR/AR geno-type at Shintoku HARC, the frequencies ofscrapie-resistant PrP genotypes in lams sig-nificantly increased compared to those beforethe selective breeding. Three years after theinitiation, the ratio of lams carrying AQ/AR orAR/AR genotypes was shifted up from４７．９to８０．０％．These results demonstrated that thebreeding with rams selected by PrP genotypes,without selection of ewes, was efficient in in-creasing the ratio of scrapie-resistant sheepin the flocks during short periods. The resultsin this study will provide the useful data fortaking measures for controlling scrapie.

Jpn. J. Vet. Res. ５４（１），２００６Information７８

Analysis of suitable PrPSc fraction for process validation and the verification of its utility

Yoshiyuki Kawabata

Laboratory of Prion Diseases, School of Veterinary Medicine,Hokkaido University, Sapporo 060-0818, Japan

Process validation is one of the methodsfor evaluating the risk of pathogen contami-nation in medical and pharmaceutical prod-ucts, cosmetics, and their raw materials. Inprocess validation of the prion contamination,it is expected that the removal efficiency willvary and the results will be overestimated de-pending on physicochemical properties of ab-normal isoform prion protein（PrPSc），the ma-jor component of prion . In this study, theauthor examined preparations of suitablePrPSc fraction for process validation, and veri-fied their utility. PrPSc aggregates with smallparticle size that were not precipitated by thecentrifugation at １００，０００x g , could be ex-tracted with Sarkosyl from brain homogen-ates of mice infected with scrapie Obihirostrain and hamsters infected with scrapie２６３K strain. Such PrPSc aggregates could also beextracted from brain microsomal fraction .Thus, I attempted to verify the utility of thePrPSc preparation extracted from brain micro-somal fraction with３％Sarkosyl, using the fil-tration steps in manufacturing process of hu-man immunoglobulin product as a model .When microsomal fraction, microsomal frac-tion treated with１％Sarkosyl and the３％‐Sarkosyl extract of microsomal fraction were

spiked into PBS as PrPSc preparation, PrPSc

filtered through a０．２２µm membrane filterwith similar removal efficiency ; removal fac-tors（Rfs）were ０．６-０．８ Log１０．No differencewas observed in the removal efficiency by the０．２２µm membrane filter when the３％‐Sark-osyl extract of microsomal fraction was spikeinto immunoglobulin solution. In contrast, theRfs increased up to２．４log１０ when the immu-noglobulin solution inoculated either microso-mal fraction or１％‐Sarkosly-treated microso-mal fraction was filtrated through the０．２２µm membrane filter. PrPSc passed throughPlanova ３５N membrane with Rfs below １Log１０，when the３％‐Sarkosyl-extract of micro-somal fraction was spiked into PBS, suggest-ing this fraction contained the PrPSc aggre-gates with particle size less than３５nm. How-ever, PrPSc was not detected in the filtrates ofPlanova ３５N when the same fraction wasspiked into immunoglobulin solution . Theseresults suggested that the PrPSc fraction withsmall particle size allows us to perform moreprecise process validation and that the evalu-ation of removal efficiency will depend onproperties of the spike materials and solutionto be examined.

Jpn. J. Vet. Res. ５４（１），２００６ Information ７９

INFORMATION: Theses for the Bachelor of Veterinary Medicine

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