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Summary The Palaeognathae comprise the flightless ratites and the volant tinamous, and together with the Neognathae constitute the extant members of class Aves. It is commonly believed that Palaeognathae originated in Gondwana since most of the living species are found in the Southern Hemisphere [1–3]. However, this hypothesis has been questioned because the fossil paleognaths are mostly from the Northern Hemisphere in their earliest time (Paleocene) and possessed many putative ancestral characters [4]. Uncertainties regarding the origin and evolution of Palaeognathae stem from the difficulty in estimating their divergence times [1, 2] and their remarkable morphological convergence. Here, we recovered nuclear genome fragments from extinct elephant birds, which enabled us to reconstruct a reliable phylogenomic time tree for the Palaeognathae. Based on the tree, we identified homoplasies in morphological traits of paleognaths and reconstructed their morphology-based phylogeny including fossil species without molecular data. In contrast to the prevailing theories, the fossil paleognaths from the Northern Hemisphere were placed as the basal lineages. Combined with our stable divergence time estimates that enabled a valid argument regarding the correlation with geological events, we propose a new evolutionary scenario that contradicts the traditional view. The ancestral Palaeognathae were volant, as estimated from their molecular evolutionary rates, and originated during the Late Cretaceous in the Northern Hemisphere. They migrated to the Southern Hemisphere and speciated explosively around the Cretaceous-Paleogene boundary. They then extended their distribution to the Gondwana-derived landmasses, such as New Zealand and Madagascar, by overseas dispersal. Gigantism subsequently occurred independently on each landmass.

To determine genetic characteristics of the maternal lineage of the Kiso horse based on polymorphisms of the mitochondrial DNA D-loop region, we collected blood samples from 136 Kiso horses, 91% of the entire population, and sequenced 411 bp from 15,437 to 15,847 in the region. First of all, we estimated the demographic history; by searching homology between the obtained and known sequences using Basic Local Alignment Search Tool, by mismatch analysis to evaluate the mutation processes using Arlequin, and by building a phylogenetic tree showing the relationship of the mtDNA haplotypes for 24 horse breeds around the world using Molecular Evolutionary Genetics Analysis softwear. The results suggested that various horses that came to Japan stayed at Kiso region and became ancestors of Kiso horse and also genetically supported the theory that the Kiso horse was historically improved by other Japanese native horse breeds. Next, we analyzed the diversity of current maternal lineage by classifying the resulting sequences, and by calculating the haplotype diversity and nucleotide diversity using Arlequin. Then, we visualized the relationship among haplotypes by a median-joining network using NETWORK 4.6.0.0. The results suggested the diversity of maternal lineage in the Kiso horse was reasonably maintained. Lastly, we predicted future change of the diversity of maternal lineage in Kiso horse by assessing the regional distribution of the acquired haplotypes. The distribution suggested that diversity of maternal lineage would possibly be reducing.

In order to contribute to conservation of the endangered Kiso horse, we clarified their genetic information using 31 microsatellite DNAs, and genotyped 125 horses, 83% of the existing breed. First, we clarified the current status of the horses. The horses were confirmed to have experienced rapid loss of population causing a bottleneck, and their effective population size was much smaller than their census size. Moreover, the number of alleles (6.3), observed heterozygosity (0.674), and expected heterozygosity (0.662) were in the same range as other endangered horses all over the world. Therefore, although their inbreeding level was not so severe (F(is): -0.017), the Kiso horse is surely one of the endangered. Second, we obtained genetic information of individuals. This information allowed us to understand the genetic distance of individuals, and might help in development of a reproductive strategy concerning the genetic distance between the mating pairs. Moreover, there appeared to be 4 subpopulations of Kiso horse, and this result was in good agreement with their historical background. Third, we confirmed that the parentage test for identification using the 31 microsatellite DNAs was highly reliable (probability of exclusion: 0.999999993). This identification increases the reliability of stud certification, and is also helpful for effective management. Understanding the genetic diversity within the population and the relationships among individuals is important to ensuring effective management for maintenance of genetic variation, and this study may help in conservation of the endangered Kiso horse.

In this study, 714 cows from 26 dairy herds were reclassified as healthy or mastitic cows on the basis of long-term somatic cell count (SCC) in milk. Cows with more than three consecutive lactation records of SCC from the first or second to fifth lactation, were selected, and their BoLA-DRB3 (DRB3) alleles were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Cows with an SCC of 300 000 cells/mL in two consecutive tests or four non-consecutive tests or cows with an SCC of > 500 000 cells/mL in any one test during lactation, regardless of parity, were classified as mastitic (n=201). Mastitic cows (n=153) from another 40 herds were considered to be infected if bacteriological testing revealed mastitis pathogens in milk. Their DRB3 alleles were identified using PCR-sequence-based typing (PCR-SBT). The differences in DRB3 allelic frequencies between healthy cows and cows with various degrees of mastitis were re-investigated. Moreover, the associations of various amino acid motifs in DRB3 alleles with resistance or susceptibility to mastitis pathogens were re-examined. DRB3.2*8(DRB3*1201) and DRB3.2*16(DRB3*1501) alleles were found to be associated with susceptibility, while DRB3.2*22(DRB3*1101), DRB3.2*23(DRB3*2703), and DRB3.2*24(DRB3*0101) alleles were found to be associated with resistance.

The objective of this study was to clarify the current status of endangered Kiso horse, population statistics and biological traits, in order to take a step for the conservation by scientific approach. We surveyed 125 Kiso horses (86.2% of the whole breed), analyzed the construction of the population, and calculated the coefficient of inbreeding and effective population size. Moreover, we confirmed coat color variations and the traditional traits of the Kiso horse, and measured their height at the withers and chest circumference to clarify their physical characteristics. The population pyramid of the horses was stationary or contractive, suggesting a reduction of the population in the near future. The effective population size of the horse (47.9) suggested that the diversity was much less than their census size, and the high coefficient of inbreeding, 0.11 ± 0.07 on average, suggested that the horses were surely inbred. The horses had only 4 coat colors; bay, dark bay, buckskin dun, and chestnut, and 116 horses (92.8%) were bayish color, suggesting the fixation in their coat color. Moreover, the majority of them had dorsal stripe (83 horses; 66.4%), and the average heights at withers(131.9 ± 4.4 cm) and chest circumference (167.1 ± 10.1 cm) were not significantly different between males and females.

It has been suggested that a variety of stresses on animals may accelerate their production of superoxide. Racehorses are considered to be exposed to substantial oxide stress due to transport and exercise for training and racing. To determine the effect of exercise and transport on racehorses in terms of superoxide and antioxidative ability, changes in the superoxide-scavenging ability of equine serum were observed using electron spin resonance (ESR). Changes in the concentration of lipid peroxide, which is produced in equine serum by superoxide, were also examined. The analysis revealed that lipid peroxide concentrations increased as a result of stress from exercise and transport. On the other hand, the superoxide-scavenging ability of equine serum showed a decline during transport, which is in sharp contrast to the increase seen immediately after a race due to the severe load exerted in exercise.

The mitochondrial DNA sequence of cytochrome b gene in a Thoroughbred horse was determined. By comparing DNA sequences between the Thoroughbred and published sequence data (two horses and one Grevyi zebra), polymerase chain reaction (PCR) primers were designed for amplification of a 590 bp DNA fragment in the cytochrome b gene, and PCR-restriction fragment length polymorphism (RFLP) analysis was studied in 140 horses of six breeds using three restriction enzymes (AciI, BamHI, RsaI). Two morphs were found using each of the three enzymes. By combining three enzymes morphs, the 140 horses examined were classified into four types. Type 2 was most frequent in all breeds.

モンゴルウマ地方品種集団における遺伝的構成を明らかにすることを目的に成長ホルモン遺伝子の多型解析を行った．まず，モンゴルウマ（Mongolian horses）とサラブレッド（Thoroughbred horses），木曽馬（Japanese native horses : Kiso）およびアングロアラブ（Anglo-Arabian horses）の成長ホルモン遺伝子の塩基配列を比較したところ，99％以上の相同性および3つの塩基置換が認められた．それらの塩基置換部位について，モンゴルウマの3つの地方品種（Wuzhumuqin : WZ, Wusen : WS, Baerhu : BH）およびサラブレッドについて多型解析を行ったところ，各部位の対立遺伝子の出現頻度に有意差が認められた．これらの結果から，モンゴルウマの成長ホルモン遺伝子は各地方品種において異なる多型状態で保存されていることが明らかになった．

Holstein Cows (n = 702) from 26 dairy herds in the Tama area of Tokyo, Japan were examined for polymorphisms of the BoLA-DRB3 allele using a PCR-RFLP method. Twenty alleles were observed and allelic frequencies ranged from < 1% to 20.3%. Nine alleles (DRB3.2*24,*16,*8,*23,*22,*3,*11, *10 and *7 in order) constituted 90.0% of all alleles. Somatic cell counts (SCC) were used to classify healthy (group 1), mastitis (group 2) and suspected (group 3) cows. Frequencies of DRB3.2*11 and DRB3.2*23 were slightly higher in group 1 than in group 2, whereas, frequencies of DRB3.2*8 and DRB3.2*16 were slightly higher in group 2 than in group 1. However, none of the differences in frequencies between the two groups were statistically significant. For combinations of alleles, frequencies of DRB3.2*8/*23 (P

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 ± 0.053 (mean ± SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.

Gamma-hydroxybutyric (GHB) acid, which is becoming popular as a drug of abuse, was shown by means of gas chromatography-mass spectrometry (GC-MS) to increase in mouse liver with time after death. The amount detected was 0.8 +/- 1.0 microg/g at 3 h after death, 4.7 +/- 1.5 microg/g at 24 h, and 8.8 +/- 0.8 microg/g at 72 h. Furthermore, GHB was detected in samples from deceased persons, at concentrations of 2.6-12.0 microg/g in liver, 0.4-7.3 microg/ml in blood, and 0-2.6 microg/ml in urine, but was not detected in the blood and urine of living persons. Although 1,4-butanediol has been suggested to be a precursor of GHB produced after death, 1,4-butanediol was not detected in any of our samples. Additionally, succinate semialdehyde arising from gamma-aminobutyric acid (GABA) transamination to GHB was also barely detectable in any of our samples. This study supports previous reports that GHB is a product of postmortem decomposition. Production of GHB increases with time after death in postmortem liver; since we were unable to identify endogenous 1,4-butanediol and succinate semialdehyde in our samples, the pathway of GHB production after death remains unclear.

Growth/Differentiation Factor 8, or GDF-8, is a member of the TGF-β family and is expressed specifically in myogenic precursor cells of the myotome during development as well as in adult axial and paraxial muscles of the mouse. It acts as a negative regulator of skeletal muscle mass, and is known as myostatin. As a part of our studies to elucidate mechanisms underlying the growth of skeletal muscle mass due to physical training in mammals including horses and humans, we isolated and cloned myostatin cDNA from equine (Thoroughbred) skeletal muscles by an RT-PCR method. The base sequence of the entire coding region of equine myostatin cDNA (DDBJ accession no. AB033541) exhibited a high degree of homology to those of other species so far reported. In the deduced amino acid sequence, 4 amino acid positions were unique to the equine myostatin, i. e., Val15 instead of Met, Leu18 instead of Val or Ala, Ala201 instead of Thr, Arg244 instead of Gly or Glu in other species. Western blot analysis of the equine skeletal muscles using anti-myostatin antibody, yielded 3 immunoreactive bands, i. e., 26, 45 and 50 kDa. The band at approximately 26 kDa probably represents mature myostatin and another at approximately 50 kDa corresponds in size to the unprocessed precursor myostatin proteins reported in the literature. Western blot analysis of equine sera of 2 independent breeds, Thoroughbred and Kiso-uma, revealed the possible presence of precursor myostatin proteins at relatively high levels. The molecular identity and the biological significance of serum myostatin immunoreactivities remain to be clarified.

Melanoma, a kind of carcinoma cutaneum, occurs at high frequency in aged gray horses. For the basic study of horse melanoma occurrence, the establishment of melanoma culture cell lines derived from three aged gray horses was attempted. Three types of enzymes, collagenase, hyaluronidase and pronase, were used for the separation of melanoma cells in the primary culture. Those enzyme treatments were highly efficient for the resolution of connective tissue. The culture medium was Iscove's modified Dulbecco's medium (IMDM) + 10% fetal calf serum (FCS; Toyobo) and the culture condition was 37°C temperature, 100% humidity, 5% CO2 and 95% air. After cloning a separated cell, three cell lines term culture were established for long term culture. These culture cell lines were named EMT-1, EMT-2 and EMT-3, respectively. These cell lines had high growth ability and the colony of the cell line was formed by a low density culture. The form of each culture cell, the cell surface and the distribution of the cytoskelton completely agreed with the characteristics observed in cancer cells. The numbers of chromosomes in these culture cell lines showed wide distribution mainly on 2n=64, and the hereditary characteristics were different from normal cell lines. Lactate dehydrogenase (LDH), nucleoside phoshorylase (NP) and glucose-6-phosphate dehydrogenase (G6PD) isozyme patterns of these culture cell lines were compared by starch gel electrophoresis, but there was no difference from the original cells. As a result of the above, it is considered that there was no contamination between the culture cell lines and the culture cells that came from the horse melanoma which showed signs of special hereditary variation.

ウマ胎児(雌)の歯根部組織を材料としてcDNAライブラリーを調製し,ウマのアメロゲニン遺伝子の塩基配列とアミノ酸配列の構造解析を行ったところ,ウマのX染色体上のアメロゲニン遺伝子cDNAの第1から第6エクソンまでのアミノ酸残基および非翻訳領域を含む696bpを解読し,Y染色体では第5エクソン領域内の24bpが欠損していることを見い出した.そこで,このY染色体の欠損部位を含む第5エクソン領域内の部分配列を増幅するためのプライマーを合成し,PCR増幅した産物をアガロースゲル電気泳動により分離したところ,雌雄間で分子サイズによる差異が認められた.この塩基配列差異による多型性を指標として,ウマの14品種個体,性染色体異常馬および培養細胞について性別判定法を行ったところ,いずれも正確な性別を判定することができた.さらに,本法によってウマの各精子についてのXまたはY精子確認を行うことができた.

Employing a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques, the complete coding sequence of cDNA for the equine SRY gene was determined. We also attempted to make clear whether the equine SRY gene transcript is expressed in the adult testis, and whether the type of transcript is expressed as linear or circular RNA. As a result, in total a 1420 bp cDNA sequence was determined. Accomplishment of 3' RACE infers that equine SRY gene was expressed as a linear RNA transcript in testicular tissue just after puberty, in contrast to the situation in mice.

The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acids. Further, the expression of Cu/Zn-SOD and Mn-SOD genes were confirmed in the equine tissues by RT-PCR and in situ hybridization.

Satellite cells from equine soleus muscles were cultured and the expression of desmin, one of the markers of skeletal muscle cells, was examined. The presence of desmin was detected in some populations of mononucleated cells on day 3 of culture, indicating that equine satellite cells were successfully isolated. On day 5, desmin was localized at the edges of the elongated myotubes but when the equine myotubes matured, no particular localization of desmin expressed diffusely within the cells was seen. Although the significance of such changes in the localization of desmin within the differentiating equine skeletal muscle cells is unknown, these results suggested that desmin may be involved in the growth and development of skeletal muscle in this species.

Complementary DNA (cDNA) clones encoding equine testicular steroidogenic enzyme 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) have been isolated from equine testicular cDNA library. Polymerase chain reaction (PCR) with 3β-HSD specific primers was performed with equine testicular cDNA, and PCR fragment was used as a probe for screening of equine testicular λ ZAP II phage cDNA library. Positive clones were excised into plasmid vector pBluescript and determined their nucleotide sequences. Total 1651 bp of the cDNA sequence was consisted with 112 bp of 5' flanking untranslated sequence, 1122 bp open reading frame encodes 373 amino acids, and putative poly-adenylation signal “AATAAA” at 385 bp downstream of the stop codon. The nucleotide and the deduced amino acid sequence of equine 3β-HSD were very similar to the known mammalian 3β-HSD sequences. From the deduced amino acid sequence, equine testicular 3β-HSD may be concluded to catalyze 3β-dehydrogenation and Δ-isomerization, but 3-ketoreduction as well as murine isoforms. As the clone was used in Northern blot analysis of equine tissues, the expression of equine 3β-HSD mRNA was prominent in the classical steroidogenic tissues including placenta.

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.

A partial cDNA fragment of equine (Thoroughbred) c-ski gene (exon 1) was cloned by means of the polymerase chain reaction (PCR) amplification, and its nucleotide sequence was determined. The nucleotide and the deduced amino acid sequences of equine c-ski showed very high homology (>90%) with human and mouse c-ski, suggesting that this gene is highly conserved in different species of mammals. Genomic DNAs from other breeds of horse, e.g., Breton, New Zealand Pony, Shire, Anglo-Arab and Tokara Horse, were also subjected to PCR analysis. No difference, however, was observed in the nucleotide sequences of the partial c-ski DNA fragments examined. The patterns of c-ski gene expression in various male fetal equine tissues, i.e., cerebrum, lung, heart, skeletal muscles etc., obtained on day 180 of pregnancy, were analyzed by means of reverse transcriptase-mediated PCR (RT-PCR). In all the tissues examined, the expression of c-ski genes was confirmed. Northern blot analysis was carried out to assess the relative expression levels of c-ski genes in the male fetal equine tissues. In all tissues examined, two forms of the transcripts were detected at molecular sizes of 7.8 kb and 6 kb, respectively. In the cerebellum and the lung, the relative expression levels were high. The expression levels in skeletal muscles were moderate compared to the other tissues. The small population size of satellite cells may explain the relatively low expression levels of c-ski genes in the well developed skeletal muscles.

To establish blood and biochemical references for the endangered Kiso horse, blood samples were collected from 111 adult Kiso horses, 74.5% of the existing breed. The samples were analyzed for 23 hematological and biochemical parameters to determine their means and standard deviations (SD). We compared the mean ± 2SD with the reference values cited in one of the most commonly used veterinary textbooks in Japan. The hematology of Kiso horses is characterized by lower erythrocyte count and hematocrit and hemoglobin levels. In addition, their serum biochemistry showed lower levels of aspartate transaminase, alkaline phosphatase, and γ-glutamyl transferase. Whether these propensities are attributed to breed-specific factors or are acquired factors remains unclear. Nevertheless, this study provides useful diagnostic indices for the endangered Kiso horse.

In this study, 714 cows from 26 dairy herds were reclassified as healthy or mastitic cows on the basis of long-term somatic cell count (SCC) in milk. Cows with more than three consecutive lactation records of SCC from the first or second to fifth lactation, were selected, and their BoLA-DRB3 (DRB3) alleles were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Cows with an SCC of200 000 cells/mL in all monthly records were classified as healthy (n=91). Cows with an SCC of300 000 cells/mL in two consecutive tests or four non-consecutive tests or cows with an SCC of500 000 cells/mL in any one test during lactation, regardless of parity, were classified as mastitic (n=201). Mastitic cows (n=153) from another 40 herds were considered to be infected if bacteriological testing revealed mastitis pathogens in milk. Their DRB3 alleles were identified using PCR-sequence-based typing (PCR-SBT). The differences in DRB3 allelic frequencies between healthy cows and cows with various degrees of mastitis were re-investigated. Moreover, the associations of various amino acid motifs in DRB3 alleles with resistance or susceptibility to mastitis pathogens were re-examined. DRB3.2*8(DRB3*1201) and DRB3.2*16(DRB3*1501) alleles were found to be associated with susceptibility, while DRB3.2*22(DRB3*1101), DRB3.2*23(DRB3*2703), and DRB3.2*24(DRB3*0101) alleles were found to be associated with resistance.

Short interspersed repetitive elements (SINEs) were isolated from the equine genome and characterized. The equine SINE (ERE-1) family has several features characteristic of tRNA-derived retroposons. The five members of the equine family of SINEs are approximately 230 nucleotides in length and terminate with a sequence rich in oligo(A). They are all flanked by direct repeats at the 5′ and 3′ ends, and such repeats are the hallmarks of retroposons. In addition, the ERE-1 family has a tRNA-related region, which is similar to tRNA Ser of Drosophila (65% identity). tRNA Ser is a novel tRNA with respect to the origin of SINEs and has not previously been recognized among the twenty tRNA-derived SINEs characterized to date. The members of the ERE-1 family were found to be distributed among five species in the genus Equus , and their amplification may have contributed to the genetic variability of their hosts during evolution.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes, identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method, with resistance and susceptibility to mastitis caused by Streptococci, coagulase-negative Staphylococci, Escherichia coli and Staphylococcus aureus was investigated. Blood samples for DNA extraction were collected from 170 Holstein cows (129 mastitis and 41 healthy cows) from 5 districts in Chiba prefecture, Japan. Susceptibility or resistance to the mastitis-causing pathogens was thought to vary by the presence of amino acid substitutions at the 9, 11, 13, and 30 positions. DRB3*0101 and DRB3*1501 had amino acid motifs of Glu(9), Ser(11), Ser(13), and Tyr(30), and they were considered to have susceptibility to all 4 mastitis pathogens. In contrast, DRB3*1101 and DRB3*1401 had amino acid motifs of Gln(9), His(11), Gly(13), and His(30) in these positions, and they also had Val(86), so these alleles were considered to have resistance to Streptococcal and coagulase-negative Staphylococcal mastitis. However, in the case of Escherichia coli mastitis, amino acid substitutions at the 9, 11, 13, and 30 positions had little effect, but rather substitutions at the 47, 67 positions of pocket 7, and at the 71, 74 positions of pocket 4, Tyr(47), Ile(67), Ala(71), and Ala(74), were associated with resistance. This motif was present in DRB3*1201.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101, *1501, *1201, and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101, *1501, *1201, and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli, and DRB3*1501 might be associated with susceptibility to Escherichia coli. However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci, and DRB3*1201, with resistance to Streptococci, Escherichia coli, and Staphylococcus aureus.

低温保存したブタの臓器からミトコンドリアDNA(mtDNA)を精製し,その精製mtDNAを用いたRFLP分析を行なった.ブタの肝臓,精巣,胎盤の各臓器を各種温度条件下で保存し,これらの臓器からmtDNAを定法に従って精製したところ,採取後21日目まで-20°Cないし-80°C下で保存された各臓器からは,いずれもRFLP分析に十分な純度のmtDNAが精製され,その電気泳動像は,ocDNAとcccDNA領域を示す2本のバンドが検出された.低温保存下の物理的な切断によるRFLP分析への影響は認められなかった.肝臓10g当たりからのmtDNAの収量は約300μgであり,RFLP分析に必要な電気泳動用試料として十分な収量であった.また,各臓器におけるmtDNAの収量は,肝臓>精巣>胎盤の順に多かったが,血液からの收量は極めて少なく,RFLPの検出には不適当と思われた.なお,低温保存臓器から精製されたmtDNAの電気泳動像は,4°Cにて21日間保存された各臓器についてはバンドが検出されなかった.以上の結果にもとづき,保存した肝臓を対象として各ブタ品種が有するmtDNA型を調査したところ,ランドレース種,ハンプシャー種においていずれも同一型が検出された.また,mtDNAのRFLPは,大ヨークシャー種,中ヨークシャー種に検出されたA型およびB型に見られるように.ブタの複雑な品種成立の過程をよく反映する指標となることが示された.さらに,三元交雑種LWDの持つmtDNA型の調査によって,ブタのmtDNAが母性遺伝を示すことが確認された.

Mastitis is an inflammatory response of the mammary gland to irritation, injury, or infectious agents and is a major problem in the dairy industry. We genotyped bovine major histocompatibility complex (BoLA)-DRB3 and BoLA-DQA1 genes in 120 Holstein cattle with clinical mastitis and 85 randomly selected Holstein cattle in Japan by polymerase chain reaction-sequence-based typing. The mastitis cattle were divided into four groups according to the bacterial species that caused the mastitis (Staphylococcus aureus, Streptococci, Escherichia, and coagulase-negative staphylococci). The BoLA-DRB3 and BoLA-DQA1 heterozygosity of each group was compared with that of the control cattle, while the expected heterozygosities based on Hardy-Weinberg proportions and the observed heterozygosities for each locus were compared for each group. The Escherichia-induced and Streptococci-induced mastitis groups showed significant differences between their expected and observed heterozygosities with regard to their BoLA-DQA1 genes. No differences were observed for any group with regard to the BoLA-DRB3 genes. We then found that two BoLA-DQA1 alleles promoted susceptibility to Streptococci-induced mastitis, namely BoLA-DQA1*0101 and BoLA-DQA1*10012 and that the homozygous BoLA-DQA1*0101/0101 and BoLA-DQA1*10011/10011 genotypes promoted susceptibility to mastitis caused by Streptococci and Escherichia, respectively. This is the first report showing that heterozygosity of the BoLA-DQA1 gene is associated with resistance to mastitis progression.

We studied the superoxide-scavenging ability (SOD-like ability) of the plasma of mares and foals before and after delivery through the use of the ESR method, with which superoxide radicals (O2-) can be detected more specifically than with conventional methods. Superoxide-scavenging ability was measured in the plasma of seven mares before delivery, and in the plasma of seven pairs of mares and foals immediately after delivery and three days, seven days, and 21 days after delivery. In the foals, a significant change was observed, with the highest superoxide-scavenging ability level seen immediately after delivery, followed by a sudden decline to three days after birth, and a gradual decline to seven days after birth. In the mares, no significant change was observed between before and after delivery. It was also shown that SOD-like ability tends to be high in mares after they develop diseases. This suggests a close association between disease and superoxide-scavenging ability.

In conclusion, we could confirm the expression of CGA in the equine submandibular gland. We consider that the salivary level as well as the plasma level of the CGA-LI can be a quantitative index for monitoring the activity of the sympathetic nervous system in physical and psychological stress of race horses.

The genetic relationships of seven Japanese and four mainland-Asian horse populations, as well as two European horse populations, were estimated using data for 20 microsatellite loci. Mongolian horses showed the highest average heterozygosities (0.75-0.77) in all populations. Phylogenetic analysis showed the existence of three distinct clusters supported by high bootstrap values: the European cluster (Anglo-Arab and thoroughbreds), the Hokkaido-Kiso cluster, and the Mongolian cluster. The relationships of these clusters were consistent with their geographical distributions. Basing our assumptions on the phylogenetic tree and the genetic variation of horse populations, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The genetic relationship of Japanese horses corresponded to their geographical distribution. Microsatellite polymorphism data were shown to be useful for estimating the genetic relationships between Japanese horses and Asian horses.

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 +/- 0.053 (mean +/- SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.

The noncoding region between tRNAPro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2-4 x 10(-8) per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.

The D-loop regions in equine mitochondrial DNA were cloned from three thoroughbred horses by polymerase chain reaction (PCR). The total number of bases in the D-loop region were 1114 bp, 1115 bp and 1146 bp. The equine D-loop region is A/T rich like many other mammalian D-loops. The large central conserved sequence block and small conserved sequence blocks 1, 2 and 3, that are common to other mammals, were observed. Between conserved sequence blocks 1 and 2 there were tandem repeats of an 8 bp equine-specific sequence TGTGCACC, and the number of tandem repeats differed among individual horses. The base composition in the unit of these repeats is G/C rich as are the short repeats in the D-loops of rabbit and pig. Comparing DNA sequences between horse and other mammals, the difference in the D-loop region length is mostly due to the difference in the number of DNA sequences at both extremities. The similarities of the DNA sequences are in the middle part of the D-loop. In comparison of the sequences among three thoroughbred horses, it was determined that the region between tRNA(Pro) and the large central conserved sequence block was the richest in variation. PCR primers in the D-loop region were designed and the expected maternal inheritance was confirmed by PCR-RFLP (restriction fragment length polymorphism).

A quick method for sex determination of horses was developed. Simultaneous amplification of the equine sex-determining region of the Y chromosome gene (SRY) and amelogenin gene (AMEL) accomplished the determination of the presence of both the Y chromosome and SRY gene. In agarose gel electrophoresis, a normal stallion showed 1 SRY band and 3 AMEL (AMELX, AMELY, and AMELX/AMELY heteroduplex) bands, and a normal mare showed a single AMELX band. In XY-mares, 3 AMEL bands were detected as in a normal stallion, but no SRY band. The present method enables a quick diagnosis for XY-mare prior to cytogenetic analysis.

ブタの白血球型分類および抗血清の標準化のため,著者およびフランス人のVAYMANらが豚同種皮膚移植免疫によって分類作製した合計23種類(59例)の抗リンパ球型血清について比較同定試験を行った.抗豚リンパ球型血清はいずれも細胞毒抗体(cytotoxic antibody)であり,その抗体価は16倍-8,192倍であった.また,25例の豚リンパ球panel cellsを用いて,リンパ球細胞毒試験(lymphocytotoxity test)によってリンパ球型反応パターンを得たのち,抗血清相互の同定試験を行ったところ,N1•N2•N3•N10系の抗血清は抗SL-A4を,またN6•N7系の抗血清は抗SL-A1をそれぞれ一部共通特異性抗体として含有し,さらにN4は抗SL-A2,N8はSL-A未分類抗体(No. 1526)とほぼ同一の特異性を示す抗血清であった.しかし,N5およびN9はSL-A系抗体の17種類には適合せず,新たに分類された抗豚リンパ球型抗体と考えられる.