Genetic diversity and relationships among native Japanese horse breeds, the Japanese Thoroughbred and horses outside of Japan using genome-wide SNP data.

Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni-are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig.

Microminipigs are extremely small-sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo- and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA-defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA-1, SLA-2 and SLA-3) and two class II (SLA-DRB1 and SLA-DQB1) genes of 14 parental Microminipigs using a high-resolution nucleotide sequence-based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp-31.0 (SLA-1*1502-SLA-3*070102-SLA-2*1601) and Hp-0.37 (SLA-DRB1*0701-SLA-DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.

Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms.

A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival was characterized. The MAPK-activated kinases, the Rsks, catalyzed the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppressed BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promoted cell survival, and inhibition of CREB phosphorylation at serine 133 triggered apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes.

A rare case of fetal extensive intracranial hemorrhage and whole-cerebral hypoplasia due to latent maternal vitamin K deficiency.

We present here a late preterm infant with extensive brain lesions resulting from vitamin K deficiency. A female infant was born after 35 weeks of gestation by emergent cesarean section because of non-reassuring fetal status. Her mother had severe eating disorder and recurrent vomiting since early pregnancy. She was immediately intubated and ventilated because she was extremely pale, hypotonic, and non-reactive. Cerebral magnetic resonance imaging immediately after birth showed intraparenchymal hemorrhage in the left frontal lobe and cerebellum, marked cerebral edema, and cerebellar hypoplasia. Coagulation studies of the infant showed hepaplastin test <5%, prolonged PT and APTT, and a marked elevation of protein induced by vitamin K absence or antagonist-II. This case highlighted a potential risk of intracranial bleeding due to maternal vitamin K deficiency and difficulty in its prediction before delivery. Vitamin K supplementation to high risk mothers might be indispensable for preventing severe fetal vitamin K deficiency. Even when coagulation studies in mothers is normal, it is imperative to provide vitamin K supplementation for total protection.

GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and ß2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

Polarized thyroid cells in monolayers cultured on collagen gel: their cytoskeleton organization, iodine uptake, and resting membrane potentials.

Porcine thyroid cells were cultured on collagen gel-coated cover glasses. They were reorganized into polarized monolayer cells; the basal cell membranes were in contact with the collagen gel, and the apical ones faced the culture medium. We studied cytoskeleton organization, resting membrane potentials, and iodine uptake of these cells. The quick-freezing and deep-etching replica method provided three-dimensional images of the cytoskeleton organization. Networks of microfilaments were observed under the apical cell membrane. In the deep cytoplasm and near the basal cell membranes, intermediate filaments predominated and were interlinked with the microfilaments. When the cells were cultured in the presence of TSH, TSH induced the formation of microvilli at the apical cell membranes and the accumulation of microfilaments under these membranes; in the deep cytoplasm, the intermediate filaments were more closely interlinked with the microfilaments. The microfilaments were immunostained with antiactin antibody. Thus, collagen is a factor in determining the cell polarity, and TSH further augments polarization through reorganizing the cytoskeletons. Electrophysiological study revealed that the resting membrane potential of cells cultured in the absence of TSH was -46 mV, and that of cells cultured in the presence of TSH was -58 mV. TSH hyperpolarized resting membrane potentials. These cells took up iodine. TSH in the medium augmented this uptake. TSH augments thyroid cell polarization through reorganizing the cytoskeletons and hyperpolarizing the resting membrane potentials and enhances iodine uptake by the cells.

Resistance to apoptosis induced by microenvironmental stresses is correlated with metastatic potential in Lewis lung carcinoma.

The apoptosis-resistant phenotype of cloned high-metastatic A11 and low-metastatic P29 cells isolated from Lewis lung carcinoma was compared. The results showed that A11 cells were more resistant to apoptosis induced by microenvironmental stresses such as serum starvation, glucose deprivation and hypoxia than P29 cells as judged by viability, DNA laddering, and chromatin condensation and fragmentation. Both cell lines were insensitive to tumor necrosis factor-alpha-mediated apoptosis. P29 cells expressed a much higher level of Fas antigen on the cell surface than A11 cells. However, both cell lines were also insensitive to Fas-mediated apoptosis. The apoptosis resistant phenotype of A11 cells was associated with the expression level of caspase-3, but not with those of Bcl-2, Bcl-X(L) Bax, p27Kip1 and DAP kinase. There was no difference between A11 and P29 cells in the expression of E-cadherin, the adhesiveness to the extracellular matrix components or the expression levels of metastasis-associated genes such as c-Ha-ras, c-jun, p53 and nm23. Furthermore, A11 cells exhibited lower motile and invasive abilities than P29 cells. These results suggest that the apoptosis-resistant phenotype is an important factor for determining the metastatic ability of A11 cells. Supporting this, P29 cells became more apoptosis-resistant after treatment of the cells with dimethylsulfoxide which is reported to enhance the experimental metastatic potential of the cells.

Enhanced neuronal death from focal ischemia in AMPA-receptor transgenic mice.

Excitatory amino acid (EAA) receptors play an important role in neuronal cell death in acute cerebral ischemia. Blocking the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of EAA receptor has been shown to reduce cell death in global cerebral ischemia. However their role in focal stroke, although suggestive, has remained more contentious. To clarify this issue, we generated transgenic mice overexpressing the AMPA receptor (AMPAR) subunit GluR2-flip which would increase AMPAR-mediated currents. Excitatory neurons in these transgenic mice are thus predicted to be more susceptible than wild-type neurons to EAA (glutamate)-induced excitotoxic damage. Consistent with this prediction, cultured neurons from transgenic mice had a lower LD50 for exposure to glutamate (10(-3)-10(-5) M for 5 min) compared to wild-type neurons. Moreover, transgenic mice subjected to permanent focal ischemia of the middle cerebral artery (MCA) using the intralumenal filament model sustained larger infarctions compared to wild-type controls. Hence we have developed a genetic mouse model that demonstrates the crucial role of AMPAR containing GluR2-flip in the pathogenesis of focal hypoxic-ischemic neuronal cell death. This model will be a valuable tool in elucidating molecular mechanisms of glutamate excitotoxicity and evaluating the efficacy of glutamate receptor antagonists in attenuating post-ischemic neuronal cell death.

Self-assembly of amphiphiles into vesicles: a Brownian dynamics simulation.

We studied the vesicles of amphiphilic molecules using a Brownian dynamics simulation. An amphiphilic molecule is modeled as the rigid rod, and the hydrophobic interaction is mimicked by the local density potential of the hydrophobic particles. The amphiphilic molecules self-assemble into vesicles with bilayer structure. The vesicles are in fluid phase, and we calculated the lateral diffusion constant and the rate of the flip-flop motion of molecules in vesicles. The self-assembly kinetics into vesicles was also investigated.

Temperature-induced expression of phb genes in Escherichia coli and the effect of temperature patterns on the production of poly-3-hydroxybutyrate.

The plasmid pNDTM2 was constructed to contain the p(R)-p(L) promoter and the phbC, A, and B genes which code for PHB synthase, beta-ketothiolase, and acetoacetyl-CoA reductase, respectively. This plasmid was transformed into Escherichia coli XL1-Blue. The effect of several thermal induction patterns on the production of poly-3-hydroxybutyric acid (PHB) was investigated based on fermentor experiments. Based on the experimental results with different induction patterns, it was found that the temperature should be controlled at 34 degrees C during the initial 10 h of cultivation to promote cell growth, and then it should be increased to 40 degrees C for induction. Then the temperature should be lowered to 37 degrees C after 5 h to relieve the effect of the heat shock.

Comparison of effects of xenobiotics on extrahepatic and hepatic microsomal drug-metabolizing enzymes in mice.

The content of microsomal protein is the same in both kidneys and small intestine, corresponding to 57% of the control value expressed as 100% in the untreated liver. The contents of P450 and cytochrome b(5), and the activity of NADPH-cytochrome c reductase in the kidney were higher than those in the small intestine, which were 17%, 22% and 41% of controls, respectively, in the former and 5%, 11% and 22% of controls in the latter. As compared with similar measurements made in the liver, the activities of substrate-metabolizing enzymes in these extrahepatic organs were very low. The activities of renal aniline hydroxylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, 7-methoxycoumarin O-demethylase and benzo(a)pyrene hydroxylase were 6%, 5%, 3%, 0.6% and 0.2% of controls, respectively. The activities of these enzymes in the small intestine were lower than those in the kidney or below the limits of detection. These results suggested that isoforms or their contents of P450 responsible for these substrate biotransformations are different among liver, kidneys and small intestine. Meantime, this study showed similar significant inductions by phenobarbital and rifampin of small intestinal and hepatic microsomal drug-metabolizing enzymes. In contrast, neither phenobarbital nor rifampin was capable of increasing renal microsomal enzymes, with the exception of benzo(a)pyrene hydroxylase which was induced by rifampin. These findings indicated that both liver and small intestine, but not kidneys contain the same phenobarbital- and rifampin-inducible P450 isoforms, cytochrome b(5) and NADPH-cytochrome c reductase. In addition, CCl(4) could be bioactivated by CYP2E1 to free radicals in the kidney which caused destruction of microsomal enzymes. In mice pretreated with phenobarbital, CCl(4) also attenuated the increase in content of P450 in the small intestine, which appeared to be a result of induction by phenobarbital of CYP2E1.

[Intraoperative fluorescein angiography with an ultrasensitive imaging tube].

Fluorescein angiographic examination may be prevented by vitreous hemorrhage or opacities in vitreoretinal diseases before vitrectomy. We performed intraoperative fluorescein angiography for such cases, using a conventional light source and an ultrasensitive video camera equipped with a silicone intensifier target tube to avoid retinal phototoxicity. Intraoperative fluorescein angiograms allowed evaluation of neovascular vessels, proliferative tissues, and avascular areas associated with diabetic retinopathy. Indentation of the pars plicata enabled us to also record fluorescein images of the area. This device may aid intraoperative assessment of vitreoretinal diseases including diabetic retinopathy.

[High-speed recording of fluorescein fundus angiography using an argon laser as the illumination source].

We constructed a new fluorescein fundus angiography system using a speedy and sensitive fundus camera and an argon laser apparatus as the illumination source. The system consists of a fluorescein fundus camera, a high-speed camera, a digital recorder, an argon laser apparatus, and a cathode ray tube(CRT)monitor. The frame speed was 200 frames/s. It was possible to record high speed fluorescein angiography with lower illumination by using an argon laser as the illumination source. This system allowed us to measure the rate of blood flow in the retinal vessels because it easily detected the dye front in the retinal vessels.

[A comparative study of TTV co-infection in patients with chronic liver disease B, C and non-B non-C].

The TTV DNA were examined in patients with chronic liver diseases B (n = 35), C (n = 44) and non-B non-C (n = 19). The clinical background, liver function and liver histological finding were compared in patients with or without TTV infection. The prevalence of TTV in patients with chronic liver diseases B, C and non-B non-C were 37.1%, 27.3% and 52.6%, respectively. There was no significant difference in liver function test between TTV positive and negative patients with chronic liver diseases B and C. The gamma-GTP level of TTV positive patients were significantly higher than that of TTV negative patients with chronic liver diseases non-B non-C. The histological findings were similar between patients with or without TTV infection. We concluded that even though the co-infection of TTV in patients with chronic liver diseases B and C was high, TTV does not act as a liver injury agent. The relationship between TTV and non-B non-C chronic liver diseases is unclear.

Ocular volumetry using fast high-resolution MRI during visual fixation.

BACKGROUND AND PURPOSE: Volumetry may be useful for evaluating treatment response and prognosis of intraocular lesions. Phantom, volunteer, and patient studies were performed to determine whether ocular MR volumetry is reproducible. MATERIALS AND METHODS: Half-Fourier single-shot RARE and FSPGR sequences at 1.5T with a 76-mm-diameter surface coil were optimized to obtain still ocular images. Volumetry accuracies of each sequence were compared with simulated subretinal phantom volumes. Ocular volumetry was performed in 15 volunteers twice in 1 week by using contiguous axial images of the globes while the subjects stared at a target, and images were acquired in 2 seconds before the subjects were instructed to blink, with this process repeated as necessary. Imaging, intraobserver, and interobserver reproducibility for volumes of the whole eyeball and anterior chamber were assessed. Ocular volumetry was also performed in 6 patients with intraocular tumors before and after treatment. RESULTS: The phantom study demonstrated that measurement error rates with RARE were significantly lower than with FSPGR (P<.01). The volunteer study demonstrated excellent imaging and intraobserver reproducibility of RARE volumetry for whole eyeballs and anterior chambers (P<.01). Although no interobserver differences were observed in anterior chamber volume measurement (P=.33), there was a significant difference between the 2 observers in eyeball volume measurement (P<.01). Follow-up volumetric data were useful for treatment decisions in all patients. CONCLUSIONS: Ocular volumetry from contiguous ultrafast RARE images obtained during visual fixation is feasible in volunteer and patient studies and is superior to FSPGR images.

Gadolinium enhancement of cauda equina after combined spinal-epidural anaesthesia.

The occurrence of neurological symptoms after spinal anaesthesia has been reported with several local anaesthetics including lidocaine, prilocaine, mepivacaine, tetracaine and bupivacaine. Although hyperbaric bupivacaine is known to induce neurological symptoms less frequently than lidocaine, a few cases of cauda equina syndrome (CES) following the intraspinal injection of bupivacaine have been reported in the English literature. We describe lumbar MRI findings for a 29-year-old woman presenting with CES after caesarean section.

Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations.

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.

Case report. Primary hypothalamic third ventriclular Burkitt's lymphoma: a case report with emphasis on differential diagnosis.

A patient with primary Burkitt-type lymphoma of the central nervous system is presented. A hypothalamic-third ventricular tumour in a man 71 years of age was diagnosed histologically as Burkitt's lymphoma. Primary Burkitt's lymphoma of the hypothalamic region is extremely rare and has not been previously reported in adults.