Decreased genetic diversity in Kiso horses revealed through annual microsatellite genotyping.

The Kiso horse is native to Japan and is on the verge of extinction. Here, we used microsatellites to characterize changes in their genetic diversity over time. We divided a population of Kiso horses that genotyped during 2007-2017 into three groups based on birth year: Group 1, 1980-1998 (70 horses); Group 2, 1999-2007 (61 horses); and Group 3, 2008-2017 (42 horses). We genotyped 31 microsatellites to calculate average number of alleles, observed heterozygosity, and expected heterozygosity. All indicators decreased across age groups. The results indicate that Kiso horses have been experiencing a drop in genetic diversity, and the population is expected to experience further decline unless appropriate measures are implemented.

Natural anti-insulin autoantibodies in cats: enzyme-linked immunosorbent assay for the determination of plasma anti-insulin IgG and its concentrations in domestic cats.

Anti-insulin immunoglobulin G (IgG) has been found in the sera of healthy cats. To determine the concentrations of these antibodies, an enzyme-linked immunosorbent assay (ELISA) for anti-insulin IgG was developed. ELISA maintained the linearity of a standard concentration line between 67.5 and 2160 ng/ml. The coefficients of variances (CVs) of intra-assays in two different plasma samples were 4.0% and 3.7%, respectively. The inter-assay CVs in two different plasma samples were 5.1% and 6.9%, respectively. The dilution curves of two samples were rectilinear. Anti-insulin IgG was detected in all 84 of the healthy cats that were tested. Plasma anti-insulin IgG concentrations ranged from 80 to 1578 µg/ml, with a median concentration of 221 µg/ml, and this value correlated positively with total plasma IgG concentrations (r=0.383, p<0.01). In an intravenous glucose tolerance test, plasma anti-insulin IgG concentrations did not alter, even with changes in plasma glucose and insulin concentrations. The ELISA that was developed was able to determine plasma anti-insulin IgG in domestic cats, and confirmed that all healthy cats had plasma anti-insulin IgG. Determining the plasma concentrations of anti-insulin IgG in cats with various pathological conditions might clarify the role of anti-insulin IgG.

Time-resolved electron paramagnetic resonance and phosphorescence studies of the lowest excited triplet states of Rh(III) corrole complexes.

The lowest excited triplet (T(1)) ππ* states of gallium (Ga) and various rhodium (Rh) 5,10,15-trispentafluorophenyl corroles (Cors) were studied in the liquid crystal (LC) E-7 and in rigid glasses by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. The triplet sublevel energies were experimentally determined by the alignment of the molecules in the LC and by magnetophotoselection in the glass. The sublevel scheme of GaCor was determined by calculating the zero field splitting (ZFS) parameters. Axial ligand effects and quantum chemical calculations were used for the sublevel assignment of RhCors. The anisotropic EPR parameters were used to determine the important higher excited states and the magnitudes of their spin-orbit coupling (SOC) contributions were evaluated. On the basis of these results and analyses, the EPR parameters and triplet lifetime were discussed for each RhCor complex.

A 9 GHz EPR imager for thin materials: application to surface detection.

A 9 GHz EPR imager that is capable of the imaging surface area of thin materials has been built. The EPR imager resolved samples spaced 1 mm apart. The developed TE(111) cavity was able to detect easily ~1.0 mM aqueous TEMPOL solution in ~1.0 mm (i.d.) glass capillary placed just above the cavity. The sensitivity measured using the TEMPOL solution showed ~0.3 of that for the modified JEOL cavity, which was in a qualitative agreement with the calculations considering the difference in the filling factors of the cavities. The relatively low measured sensitivity of the TE(111) cavity is due to utilization of the microwave field from ~3 mm aperture (hole) in the cavity wall. More importantly, the TE(111) cavity does not require inserting the sample into the cavity and placing samples into EPR tubes.

Insulin responses to administrations of amino acids and fatty acids in healthy cats.

In order to compare the stimulation ability of insulin secretion, we determined changes in plasma glucose and insulin concentrations after intravenous administration of various amino acids and essential fatty acids in clinically healthy adult cats. Plasma glucose concentrations were within the normal ranges after injection of amino acids and fatty acids. Plasma insulin concentrations increased rapidly 2 to 4 min after injection of arginine, then decreased to the basal levels at 20 min in all five cats. Insulin peak responses were significantly greater in arginine injections than in normal saline (P<0.01). Areas under the curve (AUC) of plasma insulin concentrations from 0 to 10 min after injection of arginine were significantly larger than after injection of normal saline (P<0.01) and glucose (P<0.05). Increases in AUC of plasma insulin concentration from 0 to 60 min were observed after injection of arginine, leucine, alanine, and fat emulsion. Arginine had a strong insulinotropic effect, and leucine, alanine, and fatty acids had weak ones. Besides, valine, methionine, taurine and glutamine had no stimulant activity of insulin. Given the risk of glucose toxication and required time for testing, the intravenous arginine tolerance test may be useful for estimation of insulin responses in cats.

Proton-coupled electron-transfer processes in photosystem II probed by highly resolved g-anisotropy of redox-active tyrosine YZ.

The oxidation of a redox-active tyrosine residue Y(Z) in photosystem II (PSII) is coupled with proton transfer to a hydrogen-bonded D1-His190 residue. Because of the apparent proximity of Y(Z) to the water-oxidizing complex and its redox activity, it is believed that Y(Z) plays a significant role in water oxidation in PSII. We investigated the g-anisotropy of the tyrosine radical Y(Z)(•) to provide insight into the mechanism of Y(Z)(•) proton-coupled electron transfer in Mn-depleted PSII. The anisotropy was highly resolved by electron paramagnetic resonance spectroscopy at the W-band (94.9 GHz) using PSII single crystals. The g(X)-component along the phenolic C-O bond of Y(Z)(•) was calculated by density functional theory (DFT). It was concluded from the highly resolved g-anisotropy that Y(Z) loses a phenol proton to D1-His190 upon tyrosine oxidation, and D1-His190 redonates the same proton back to Y(Z)(•) upon reduction.

Time-resolved high-frequency EPR studies on magnesium and zinc tetraphenylporphines in their lowest excited triplet states.

The lowest excited triplet (T(1)) states of magnesium and zinc tetraphenylporphines (MgTPP and ZnTPP) were studied by time-resolved (TR) high-frequency/high-field W-band electron paramagnetic resonance (hf-EPR) spectroscopy in rigid glasses at low temperatures. Inspections of the TR-hf-EPR spectra of the spin-polarized triplets revealed that the zero field splitting (ZFS) parameters, D and E, for MgTPP and ZnTPP triplets were nearly the same. At the same time, their g-tensors were found to be different. These results are interpreted quantitatively in terms of spin-orbit couplings (SOCs) and angular momenta among the excited states, giving a magnitude of SOC in the T(1) state of ZnTPP. For the first time, both the TR-hf-EPR spectra and corresponding time profiles were acquired on the ZnTPP's triplet at room temperature in liquid paraffin solution with the populations of the electron spin states being in Boltzmann equilibrium. Because of relatively fast paramagnetic relaxation in rotating triplet at room temperature, the spectra and time profiles were free from the effects of microwave saturation that allowed for the direct measurement of the absolute intersystem crossing ratios P(x):P(y):P(z) 0.085:0.085:0.83. All of these results have demonstrated advantages and new perspectives of the W-band EPR spectroscopy.

Plasma leptin concentration in dogs with diabetes mellitus.

The plasma leptin concentration was evaluated in dogs with diabetes mellitus. Twenty normal and sixteen diabetic dogs were divided into nonobese and obese groups based on body condition score, respectively. The obese normal dogs had significantly higher plasma leptin concentrations than the nonobese normal dogs, whereas there was no significant difference between the nonobese and obese diabetic dogs. In addition, the plasma leptin concentration in the obese diabetic dogs was significantly lower than that in the obese normal dogs. In conclusion, the plasma leptin concentrations in the diabetic dogs were affected by factors other than adiposity.

Thymic hypoplasia in Japanese black calves with stillbirth/perinatal weak calf syndrome.

To investigate the relationship between the decreased immunity associated with infirmity and low body weight at birth as a consequence of intrauterine growth retardation in Japanese Black calves with stillbirth/perinatal weak calf syndrome, the thymuses and spleens of 13 calves with this syndrome, weighing less than 20 kg at birth, were examined histopathologically. Cytokeratin staining of the thymus was also carried out to examine its composition. The thymus and spleen were classified as grades 0-4 and I-III according to their hypoplasia, respectively. All calves showed a decreased number of thymocytes. One calf was classified as grade 1, which was characterized by a starry sky appearance. Five calves were classified as grade 2, demonstrating a reversion of the cortex-to-medulla ratio, and the rest were classified as grades 3 and 4 showing an indistinguishable boundary between the cortex and medulla. The thymuses of grade 3 and 4 were occupied by stroma cells, and their Hassall bodies and other structures were rarely observed. Six of 13 calves showed a decreased number of splenocytes, grade II or III, and their red and white pulp regions were unclear. The intrauterine growth retardation caused by lack of growth factors during the fetal period might have induced thymic hypoplasia associated with decreased immunity in the calves with stillbirth/perinatal weak calf syndrome. Therefore, intrauterine growth retardation might be associated with one of the causes of decreased immunity involved in infirmity in this syndrome.

Computer tomography diagnosis of meningoencephalocele in a calf.

A 24-day-old female Holstein calf had a soft, painless fluctuating swelling on the median plane in the frontal region, but did not show any clinical symptoms including neurological signs. Computer tomography (CT) distinctly showed the cyst filled with fluid and part of the encephalon. Hence, this swelling was diagnosed as meningoencephalocele, but not meningocele. The meningoencephalocele was successfully repaired surgically. Meningoencephalocele can thus be easily recognised by CT in a calf.

Use of urine albumin/creatinine ratio for estimation of proteinuria in cats and dogs.

The clinical utility of the urine albumin/creatinine ratio (UAC) using a simplified analyzer for estimation of proteinuria was studied in cats and dogs. Measurement results for diluted feline and canine albumin standard solutions showed linearity. Although conversion formulas (y=1.28x+1.04 and y=1.67x+10.47 for cats and dogs, respectively) were necessary, urine albumin concentrations could be determined in both animals. In cats and dogs with proteinuria, the UAC changed parallel with the urine protein/ creatinine ratio (UPC), and the Log UAC and Log UPC were significantly correlated (r=0.803 (p<0.01) in cats, r=0.801 (p<0.01) in dogs). The UAC using an UAC analyzer could be used clinically as one of the basic in-hospital laboratory tests for estimation of proteinuria in cats and dogs.

Peculiarity in the electronic structure of Cu(II) complex ferromagnetically coupled with bisimino nitroxides.

By means of the electron spin resonance (ESR) technique, we have investigated the electronic structures of the tridentate imino nitroxyl diradical complex with copper(II) (Cu-bisimpy), which has a square planar structure and a ground quartet state with an extremely strong ferromagnetic exchange interaction, and its related compounds (bisimpy = 2,6-bis(1'-oxyl-4',4',5',5'-tetramethyl-4',5'-dihydro-1' H-imidazol-2'-yl)pyridine). It was clarified that Cu-bisimpy had unique magnetic orbitals, compared with the biradical ligand (bisimpy), a zinc(II) biradical complex (Zn-bisimpy) and a copper(II) terpyridine complex (Cu-tpy) (tpy = 2,2';6',2''-terpyridine). Multifrequency ESR spectroscopy provided a reliable set of magnetic parameters of Cu-bisimpy, which has a small g anisotropy ( g x = 2.02, g y = 2.01, g z = 2.08) and small hyperfine coupling with Cu (|A x| = 42.0 MHz, |A y|

Development of a control system for pulsed-electron spin resonance spectrometers.

A pulse control and data acquisition system with high time resolution of 300 ps and data-averaging rate of 100 kHz for a pulsed-ESR spectrometer was constructed. The system consists of two sets of 3.34 GHz data timing generator and a dual-channel analog-to-digital converter with 1 GHz samples, both of which are commercially available. We developed a scheme and software to control these equipments with high efficiency in pulsing design, data acquisition, and accurate synchronization of the devices. The system was developed at Ku band (17.5 GHz) and demonstrated some examples of electron spin echo and double quantum measurements.

Pedigree analysis of factor XI deficiency in Japanese black cattle.

Using a DNA-based diagnostic test for factor XI deficiency in Japanese black cattle, we surveyed 123 cattle (42 sires and 81 dams) in Gifu and Hyogo prefectures, and calculated gene frequencies. In sires, we drew up the pedigree network of the cattle with the factor XI deficiency. Results showed that the mutated allele of factor XI deficiency was retroactive in at least 6 or more generations of sires. Frequencies of the mutant gene were higher at 26.4% in total, and at 33.3% in sires. All 7 cattle with the homozygote of mutated allele were clinically normal, and showed no bleeding episodes. The mutated allele of factor XI deficiency might be widespread among Japanese black cattle.

A new pulse width reduction technique for pulsed electron paramagnetic resonance spectroscopy.

We present a new technique for a microwave pulse modulator that generates a short microwave pulse of approximately 1ns for use in an electron paramagnetic resonance (EPR) spectrometer. A quadruple-frequency multiplier that generates a signal of 16-20GHz from an input of 4-5GHz was employed to reduce the rise and fall times of the pulse prepared by a PIN diode switch. We examined the transient response characteristics of a commercial frequency multiplier and found that the device can function as a multiplier for pulsed signal even though it was designed for continuous wave operation. We applied the technique to a Ku band pulsed EPR spectrometer and successfully observed a spin echo signal with a broad excitation bandwidth of approximately 1.6mT using 80 degrees pulses of 1.5ns.

Expression analysis of CCL27 and CCL28 mRNA in lesional and non-lesional skin of dogs with atopic dermatitis.

Chemokines are important regulators of the selective recruitment of inflammatory cells into sites of allergic inflammation. Since canine atopic dermatitis (AD) shares many clinical features of human AD, patterns of chemokine production in dogs may also be similar with those in humans. The aim of this study was to examine mRNA expression of CCL27 and CCL28 in lesional skin of dogs with AD to demonstrate similarity of chemokine production with human counterparts. RNA was extracted from skin biopsy specimens of 12 dogs with AD. The mRNA expression of CC chemokines (CCL4, CCL19, CCL20, CCL21, CCL24, CCL27 and CCL28) was analyzed by quantitative real-time PCR and was compared between lesional and non-lesional skin. Seven types of chemokines examined were constitutively expressed in both lesional and non-lesional skin. It was found that mRNA expression levels of CCL27 and CCL28 among the chemokines were significantly different between lesional and non-lesional skin (P<0.05). Expression level of CCL27 mRNA in lesional skin was significantly lower than that in non-lesional skin. On the other hand, CCL28 mRNA expression in lesional skin was found to be higher than that in non-lesional skin. These results suggest that CCL28 but not CCL27 may play important roles in immunopathogenesis of canine AD, indicating that experimental canine study may provide additional information that can be extrapolated to human AD.

Assignment of the SLA alleles and reproductive potential of selective breeding Duroc pig lines.

BACKGROUND: Pigs with defined swine leukocyte antigen (SLA) haplotypes and their detailed information are useful for transplantation and immunological studies. We developed two herds of SLA homozygous Duroc pigs with novel SLA haplotypes and characterized their reproductive potential. METHODS: For selective inbreeding, a pair of Duroc pigs was chosen as initial breeders, and substantial breeding within progenies was carried out for eight generations. In the selective breeding Duroc pigs, SLA haplotypes were assigned by nucleotide sequence determination of reverse transcription polymerase chain reaction (RT-PCR) products of three SLA classical class I genes and two class II genes. Based on this sequence information, we developed a rapid and simple SLA class II DNA typing method by polymerase chain reaction-sequence specific primer (PCR-SSP) technique. As a complementary method for the characterization of the SLA haplotypes, genetic polymorphisms of 36 microsatellite (MS) markers within the SLA region were also analyzed in the selective breeding pigs with SLA homozygous/heterozygous haplotypes. RESULTS: Among the selective breeding pigs from the third to fifth generations, only two SLA haplotypes were identified by the RT-PCR based SLA typing method; Hp-27.30 (SLA-1*08an03, SLA-1*06an04, SLA-2*0102, SLA-3*0101 DRB1*1101 and DQB1*0503) and Hp-60.13 (SLA-1*an02, SLA-2*1002, SLA-3*0502, DRB1*0403 and DQB1*0303). In these two SLA haplotypes, two class I haplotypes, Hp-27.0 and Hp-60.0, are novel. Furthermore, two class II haplotypes, Hp-0.30 and Hp-0.13, which were previously reported in Korean native pigs and pigs of Hanford breed, respectively, were also assigned by a simple assay using a PCR-SSP technique in the entire selective breeding stock. Moreover, two haplotype specific MS patterns were observed across the entire SLA region in the selective breeding (homozygous/heterozygous) pigs. No morphological abnormalities were observed in selective breeding pigs. The theoretical inbreeding coefficient at the eighth generation was 78.5%. In all generations of selective breeding pigs, litter sizes were comparable and weaning weights from the fifth to eighth generation produced progenies significantly lighter (P < 0.01) than those in the non-selective breeding pigs. CONCLUSIONS: We established and characterized SLA homozygous Duroc herds with two kinds of haplotypes that can be used as a new resource for transplantation and other biomedical studies.

Propionate tolerance test for determination of insulin secretion in a hyperglycemic Japanese Black steer.

A propionate tolerance test (PTT) was used to determine the pathophysiology of a Japanese Black steer with hyperglycemia. In the hyperglycemic steer, a low insulin secretion was confirmed by a glucose tolerance test (GTT), so that the hyperglycemic steer was diagnosed as insulin-dependent diabetes mellitus. Although the plasma insulin concentration in the control cattle increased in response to propionate stimulation, a low insulin response to PTT was observed in the diabetic steer. The fact that both PTT and GTT determined that the diabetic steer had low insulin secretion suggests that the PTT might be an effective diagnostic tool for diabetes mellitus in cattle.

Cloning, expression and investigation for polymorphisms of canine peroxisome proliferator-activated receptors.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors implicated in lipid metabolism. In this study, the full-length cDNA of canine PPARbeta and gamma were sequenced, and expression of PPARs was evaluated in normal tissues and primary cultures of adipocytes in dogs, followed by investigations for polymorphisms of canine PPARgamma. Comparison of the deduced amino acid sequences of canine PPARbeta and gamma cDNA with that of human PPARbeta and gamma cDNA revealed 95.9% and 98.2% identity, respectively. PPARbeta expression was ubiquitous and high PPARgamma expression was detected in the subcutaneous and omental adipose tissues, spleen and large intestine. Canine PPARgamma mRNA expression in cultured adipocytes began to increase from 4 days after induction of differentiation, and increased nearly ninefold within 10 days after induction of differentiation. Although expression level of PPARalpha was low in the cultured adipocytes, it slightly increased within 10 days. In contrast, expression of PPARbeta showed only small variations during adipocyte differentiation, though expression levels were relatively high. These results suggest that PPARgamma may play an important role in adipocyte differentiation in dogs. Investigations for polymorphisms of PPARgamma revealed a silent polymorphism, C1362T, in 3 of 92 dogs.

Japanese black cattle with orotic aciduria detected by gas-chromatography/mass-spectrometry.

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.