Newly identified interferon tau-responsive Hes family BHLH transcription factor 4 and cytidine/uridine monophosphate kinase 2 genes in peripheral blood granulocytes during early pregnancy in cows.

In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.

Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals.

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.

Molecular Evidence for Convergence and Parallelism in Evolution of Complex Brains of Cephalopod Molluscs: Insights from Visual Systems.

Coleoid cephalopods show remarkable evolutionary convergence with vertebrates in their neural organization, including (1) eyes and visual system with optic lobes, (2) specialized parts of the brain controlling learning and memory, such as vertical lobes, and (3) unique vasculature supporting such complexity of the central nervous system. We performed deep sequencing of eye transcriptomes of pygmy squids (Idiosepius paradoxus) and chambered nautiluses (Nautilus pompilius) to decipher the molecular basis of convergent evolution in cephalopods. RNA-seq was complemented by in situ hybridization to localize the expression of selected genes. We found three types of genomic innovations in the evolution of complex brains: (1) recruitment of novel genes into morphogenetic pathways, (2) recombination of various coding and regulatory regions of different genes, often called "evolutionary tinkering" or "co-option", and (3) duplication and divergence of genes. Massive recruitment of novel genes occurred in the evolution of the "camera" eye from nautilus' "pinhole" eye. We also showed that the type-2 co-option of transcription factors played important roles in the evolution of the lens and visual neurons. In summary, the cephalopod convergent morphological evolution of the camera eyes was driven by a mosaic of all types of gene recruitments. In addition, our analysis revealed unexpected variations of squids' opsins, retinochromes, and arrestins, providing more detailed information, valuable for further research on intra-ocular and extra-ocular photoreception of the cephalopods.

Embryonic and paralarval development of the central nervous system of the loliginid squid Sepioteuthis lessoniana.

The embryonic development of the central nervous system (CNS) in the oval squid Sepioteuthis lessoniana is described. It has three distinct phases: (1) The ganglionic accumulation phase: Ganglionic cell clusters develop by ingression, migration, and accumulation of neuroblasts. (2) The lobe differentiation phase: Ganglia differentiate into lobes. The phase is identified by the beginning of an axogenesis. During this phase, neuropils are first formed in the suboesophageal mass, then in the basal lobe system, and finally in the inferior frontal lobes and the superior frontal-vertical lobe systems. (3) The neuropil increment phase: After the shape of the lobes reached its typical form, neuropil growth occurs, specifically in the vertical lobe. The paralarval central nervous system (CNS) is characterized by neuronal gigantism of the giant fibers and some suboesophageal commissures and connectives. The neuropil formation in the CNS of S. lessoniana occurs somewhat earlier than in Octopus vulgaris, although the principal developmental plan is quite conservative among the other coleoids investigated. Some phylogenetic aspects are discussed based on the similarities in the morphologic organization of their brains.

The determination of brain water content: microgravimetry versus drying-weighing method.

The microgravimetric technique and the drying-weighing method for the determination of brain water content are analyzed and compared. A new method has been devised for the automatic production of the gradient column. For gravimetry, tissue samples weighing more than 30 mg have proven adequate for measurement. Specific gravity (SG) should be determined as early as 1 minute after tissue is inserted into the gradient column. Calculations of cerebral blood volume (CBV) from changes in SG of both brain tissue and intravascular perfusate have shown that the SG of brain tissue is considerably influenced by changes in CBV. This is because the SG of blood is higher than that of brain tissue, and may lead to a decrease of SG of about 0.002 in anemic cortex and of 0.001 in anemic white matter, which will simulate a false increase in tissue volume as water of 4% and 2%, respectively. This methodological error may be relevant when the early stages of ischemic brain edema development are studied. Water content of brain tissue can also be determined with acceptable accuracy by vacuum freeze-drying samples of brain tissue weighing about 100 mg. In contrast to cortex, white matter shows a wide range of individual and regional differences in water content. Thus, conclusions on the presence of brain edema drawn from tissue water determinations should always be subjected to cautious analysis and criticism.

The use of three-dimensional computed tomographic angiography in the accurate diagnosis of internal carotid artery aneurysms: degree for expression of posterior communicating and anterior choroidal arteries.

We have improved the scan protocol of three-dimensional computed tomographic angiography (3D-CTA) to maximize the accuracy of examining unruptured cerebral aneurysms. Sixty-seven cases of suspected internal carotid-posterior communicating (Pcom) and internal carotid-anterior choroidal (Ant Cho) artery aneurysms were evaluated as to the opacification of arteries. In the 46 cases of suspected fetal-type Pcom and Ant Cho aneurysms, the arteries were opacified from its origin to the periphery in all cases. A final diagnosis of the presence of an aneurysm was made possible by complete opacification. A definite diagnosis was impossible to make in only ten cases having adult-type Pcom. In conclusion, 3D-CTA, using our own scan protocol, was proven to be quite useful for making a more accurate diagnosis.

Differentiation between chondroma and squamous cell carcinoma: evaluation of subtraction image of MRI.

The image processing procedure has become widely applied as a visual aid in imaging diagnosis. The subtraction image of MRI obtained by digitally subtracting an unenhanced image from a contrast-enhanced image, depicted a discrete distribution of the contrast agent. Subtraction images in the clinical cases were compared. The subtraction image in a case of chondroma demonstrated a relatively uniform distribution of a contrast agent, with a well delineated neoplastic lesion. The subtraction image in a case of squamous cell carcinoma, the contrast agent was distributed heterogeneously in the carcinoma and well permeated into the muscle. Different patterns of subtraction image were obtained in the clinical cases of chondroma and squamous cell carcinoma in this study. The findings suggest the potential usefulness of the subtraction image for diagnosing the degree of malignancy.

Application of short-time magnetic resonance examination for intervertebral disc diseases in dogs.

The usefulness of magnetic resonance (MR) is already established, but it has a disadvantage of requiring a long scanning time. A short-time examination is more or less needed so as to be more practical in veterinary clinics. A protocol of the short-time MR examination was devised based on parameters determined, and validity of the protocol was assessed through the diagnosis of clinical cases with intervertebral disc diseases. With this protocol, it was possible to complete an MR examination for the spine within 15 min. The MR images and myelographic findings were correlated well in this study, suggesting the short-time protocol of MR examination can be used in the clinical diagnosis of spinal diseases.

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs.

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.

A case of a dog with thickened calvaria with neurologic symptoms: magnetic resonance imaging (MRI) findings.

A 6-year-old female mongrel dog weighing 9.0 kg was presented ananastatic, with clouding of consciousness, bilateral loss of hearing and depressed reactivity of the eyes to light. Magnetic resonance imaging (MRI) examination showed that the calvaria was markedly thickened with compression to the cerebrum and cerebellum. The case of a dog with thickened calvaria with compression of the cerebrum and cerebellum which could not be diagnosed by conventional measures was amenable to diagnosis by MRI. With increased application of MRI examination, such canine cases might increase in number.

Investigation of the short-term effect of chemonucleolysis with chondroitinase ABC.

Chondroitinase ABC (C-ABC) is expected to be a novel agent for chemonucleolysis. The effect of C-ABC was investigated by magnetic resonance (MR) and radiograph. C-ABC was administered into the lumbar intervertebral disks on the clinically normal beagles (n=5), in a dose of 50 microl (12.5 units as C-ABC). MR scans were performed pre-dose, and 1, 3, 7, 14 and 28 days after administration of C-ABC, and the signal intensity (SI) of the nucleus pulposus was measured. Radiographs were taken pre-dose, and 1, 2, 3, 4, 5, 6, 7, 14 and 28 days post-dose, to evaluate narrowing of the disk space in terms of height index (HI). In addition, the quantity of the chondroitin sulfate (CS) and the hyaluronic acid (HA) in the nucleus pulposus were measured by high performance liquid chromatography on day 28 after dosing. SI and HI continuously decreased, following the injection to 37.1% and 78.9% of the pre-dose values, respectively. Statistically significant differences (p<0.01) were observed between the C-ABC group and the control group in the respects on day 1 post-dose. CS and HA contents of the nucleus pulposus were noted to be significantly decreased on day 28 (p<0.01) in the treated group. This agent proved to degenerate proteoglycans in the nucleus pulposus, thus progressively reducing the interdiskal pressure from day 1 post-dose onwards. It is concluded that C-ABC is expected to afford its efficacy from early in the course of chemonucleolysis.

Studies on Brugia pahangi in inbred hamsters. 1. Susceptibility of inbred GN and APG hamsters.

The susceptibility of inbred GN and APG hamsters to Brugia pahangi infection was studied. Male GN hamsters infected subcutaneously with 100 infective larvae showed 100% patency with the average prepatent period of 69 days. The average microfilarial density was 148/40 c.mm at 26 weeks and reached 1,000/40 c.mm at days 280. The rate of adult recovery was as high as 36%. Most worms were recovered from the heart and lungs (46%) and testes (40%). The male GN hamster-B. pahangi model could be a good substitution for the jird-B. pahangi model. Female GN and male APG hamsters seemed to be less susceptible than male GN hamsters. However, they could still be a better alternative to outbred hamsters due to their high rates of patency (92 and 100%), relatively high microfilarial densities (31 and 39/40 c.mm at 26 weeks) and reasonable rates of adult recovery (23.5 and 17.2%).

[Fundamental problems and improved methods in the measurement of specific gravity of cerebral tissues. (author's transl)].

Gravimetric determination of brain tissue water content through the measurement of specific gravity (SG) still poses some basic problems. We have studied some of them with the aid of a new gradient column. Using a specially designed conical cylinder to distribute the solution in a graded manner, and a floating apparatus, a gradient column was automatically prepared with a high linearity (r greater than 0.99990). Analysis of equilibration time-course curves of cat brain tissue fragments with various sizes showed that it was more accurate to use volumes as large as 50 mg, and to measure as early as one minute after tissue insertion. In normal brain, SG of grey matter was usually higher than that of white matter. In contrast, in a brain with oligemia or with low hemoglobin contents of blood, this relationship was reversed. Thus, SG can be influenced not only by water content, but also by other factors. As SG of blood is higher than that of brain tissue, cerebral blood volume (CBV) is of particular interest. Assuming the total brain volume as equal to 1, CBV and volume of brain proper excluding intravascular blood can be expressed as x and 1-x respectively. In a given brain tissue, SG of blood or perfusate a, and SG of whole brain tissue A can be measured. If SG of brain proper is expressed as y, the following equation can be introduced: ax + y(1-x)=A. If a and A are obtained from two different brains under the condition of constant CBV, x, 1-x and y can be calculated. For this purpose, values from normal brain and those from perfused brain with isotonic saline were applied. The calculated CBV was 7% in the grey and 3% in the white. SG of brain proper was as low as 0.002 in the grey and 0.001 in the white as compared to that of whole brain tissue. When these values are applied to the equation of percent change in tissue volume as water = (SG of control tissue-1)-(SG of experimental tissue-1)/(SG of experimental tissue-1) x 100%, the false positive increase of water in a completely ischemic brain without edema is approximately 4% in the grey and 2% in the white. Then, care should be taken in the assessment of water increase from SG, particularly in the early stage of ischemic edema.

Octopus gonadotrophin-releasing hormone: a multifunctional peptide in the endocrine and nervous systems of the cephalopod.

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

The interdependence of pitch and temporal judgments by absolute pitch possessors.

The auditory tau and the kappa effects show that there is time-pitch interdependence in our perception. Our judgments of pitch separation between two tones depend on the temporal interval between them (the auditory tau effect), and our judgments of the tones' temporal interval depend on their pitch separation (the kappa effect). The mechanisms underlying this interdependence were investigated by studying the auditory tau and the kappa effect in three experiments. Comparisons were made between results obtained from subjects with absolute pitch and those who did not have absolute pitch, and two frequency ranges of pure tones (octave and whole-tone conditions) were selected. The procedures had been used in previous experiments (Shigeno, 1986), in which the auditory tau and the kappa effects were compared in speech and nonspeech stimuli. The present results demonstrate that the auditory tau effect does not occur when possessors of absolute pitch judge the closeness of stimuli in pitch, except when the stimulus continuum consists of tones that do not correspond to musical notes in the whole-tone condition. The kappa effect was obtained in the judgment of possessors of absolute pitch in both the octave and the whole-tone conditions. These findings suggest that the interaction between temporal interval and pitch judgment might be explained in terms of the two different memory modes for retaining the pitch of tones, and that these effects occur at the precategorical level.

Assimilation and contrast in the phonetic perception of vowels.

The perceptual mechanisms of assimilation and contrast in the phonetic perception of vowels were investigated. In experiment 1, 14 stimulus continua were generated using an /i/-/e/-/a/ vowel continuum. They ranged from a continuum with both ends belonging to the same phonemic category in Japanese, to a continuum with both ends belonging to different phonemic categories. The AXB method was employed and the temporal position of X was changed under three conditions. In each condition ten subjects were required to judge whether X was similar to A or to B. The results demonstrated that assimilation to the temporally closer sound occurs if the phonemic categories of A and B are the same and that contrast to the temporally closer sound occurs if A and B belong to different phonemic categories. It was observed that the transition from assimilation to contrast is continuous except in the /i'/-X-/e/ condition. In experiment 2, the total duration of t 1 (between A and X) and t 2 (between X and B) was changed under five conditions. One stimulus continuum consisted of the same phonemic category in Japanese and the other consisted of different phonemic categories. Six subjects were required to make similarity judgements of X. The results demonstrated that the occurrence of assimilation and contrast to the temporally closer sound seemed to be constant under each of the five conditions. The present findings suggest that assimilation and contrast are determined by three factors: the temporal position of the three stimuli, the acoustic distance between the three stimuli on the stimulus continuum, and the phonemic categories of the three stimuli.

Effects of indomethacin on rCBF during and after focal cerebral ischemia in the cat.

The effect of indomethacin on rCBF was studied in cats anesthetized with Nembutal either under resting conditions or with temporary middle cerebral artery (MCA) occlusion. RCBF was measured by the microsphere method. In control animals (n = 3), indomethacin (4 mg/kg, i.v.) significantly reduced rCBF by about 25% in both cortex (from 44 +/- 6 to 32 +/- 3 ml/100 g/min, p less than 0.001) and white matter (from 36 +/- 4 to 26 +/- 2 ml/100 g/min, p less than 0.001). After MCA occlusion rCBF was markedly decreased in the sylvian region ipsilateral to occlusion (ischemic core) (from 38 +/- 4 to 14 +/- 2 ml/100 g/min in cortex, 4 animals). Although pretreatment with indomethacin (4 mg/kg) (4 animals) 30 min prior to occlusion did not alter rCBF during ischemia, a marked enhancement of reactive hyperemia was observed in the ischemic core immediately upon reperfusion following 2 h occlusion (54 +/- 11 untreated vs 95 +/- 13 treated, p less than 0.05). In the delayed postischemic period, namely 2 h after recirculation, rCBF still remained to be higher in the animals treated with indomethacin (40 +/- 6 untreated vs 96 +/- 9 treated, p less than 0.001). Such an effect of indomethacin for ameliorating postischemic blood flow in both the immediate and delayed period was less prominent in the adjacent area (penumbra) ipsilateral to occlusion. In the contralateral hemisphere, indomethacin caused slight reduction in rCBF during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral edema following experimental subarachnoid hemorrhage.

The development of cerebral edema after experimental subarachnoid hemorrhage (SAH) was studied in cats by determining regional brain tissue water content with the microgravimetric technique as well as the drying-weighing method. SAH was induced by withdrawing needles previously pierced into one or both infraclinoid internal carotid arteries through a unilateral transorbital approach. Serial determinations of regional cerebral blood flow (rCBF) by labelled microspheres, and monitorings of vital signs such as intracranial pressure (ICP), blood pressure and EEG were carried out up to 24 h after SAH. Animals could be classified into three grades according to the severity of SAH. In grade I, the increase of ICP was transient and minor. In grade II, ICP increased up to 200 mm Hg with a marked reduction of rCBF below 20% of control in cerebral hemispheres. Following subsequent reduction of ICP, rCBF increased over control, indicating reactive hyperemia. Thereafter, a great reduction of rCBF was again observed. In grade III, rCBF was sustained at essentially zero flow with the presence of continuously increased ICP above 100 mm Hg. Cerebral edema was observed particularly in the parasagittal water-shed areas of all grade II animals. It is concluded that cerebral edema complicating SAH is caused by the combination of an initially induced global cerebral ischemia and the subsequent recovery of cerebral circulation. Post SAH hypertension is another factor to exacerbate the development of cerebral edema.