Identification and characterization of novel developmentally regulated neural-specific proteins, BRINP family.

Processes of neuronal differentiation involve activation of a set of neuronal specific genes and cessation of cell proliferation in postmitotic neurons. Previous studies revealed that bone morphogenetic protein (BMP) and retinoic acid (RA) play important roles in the differentiation of peripheral sympathetic neurons such as the synergistic induction of responsiveness to specific neurotrophic factors. In the present study, while trying to clarify the mechanism of the BMP/RA-actions, we identified a novel neural-specific protein, BMP/RA-inducible neural-specific protein-1 (BRINP1) which shows no similarity to other known proteins. Subsequently, two homologous proteins, BRINP2 and BRINP3, making up the BRINP family, are identified. Individual BRINP genes have distinct regulatory mechanisms of expression within the nervous system. In rodent brain, BRINP1 is expressed from earlier developmental stage, i.e. E9.5, and widely expressed in various neuronal layers and nuclei of the adult animal, while BRINP2 and BRINP3 were detectable from E11.5 and expressed in rather limited regions in a complementary manner. During the course of perinatal development of sympathetic neurons, BRINP1 is induced from earlier embryonic stage and further increased toward adult stage, while BRINP3 expressed from earlier stage is replaced by BRINP2 expression which increases postnatally in accordance with the action of BMP2 and RA. Furthermore, when expressed in nonneuronal cells, all three BRINP family proteins suppressed the cell cycle progression. Possible physiological functions of BRINP family members in the development of the nervous system are discussed.

Histological changes in monoaminergic neurons of Long-Evans Cinnamon rats.

The Long-Evans Cinnamon rat, an animal model of Wilson's disease, is an inbred mutant strain with spontaneous hepatitis isolated from Long-Evans rats. The copper concentration in the brains of Long-Evans Cinnamon rats at 4 weeks of age was lower than that of controls, but higher than that of controls at 20 weeks of age. We investigated the tyrosine hydroxylase and 5-hydroxytryptamine immunoreactive fiber densities in the brains of Long-Evans Cinnamon rats aged 4, 10, and 20 weeks by immunohistochemistry, comparing them with Long-Evans Agouti rats used as controls. Tyrosine hydroxylase immunoreactive fiber densities in the cingulate cortex, hippocampus and cerebellum in Long-Evans Cinnamon rats were significantly lower than those of Long-Evans Agouti rats at 4 and 10 weeks of age. On the other hand, 5-hydroxytryptamine immunoreactive fiber densities in the cingulate cortex, caudate-putamen, hypothalamus, and hippocampus in Long-Evans Cinnamon rats were significantly higher than those of controls at 4, 10 and 20 weeks of age. In the cingulate cortex and caudate-putamen, 5-hydroxytryptamine immunoreactive fiber densities became gradually higher with age. The number of aberrant 5-hydroxytryptamine immunoreactive fibers in the cingulate cortex, caudate-putamen, hypothalamus and hippocampus in LEC rats was significantly higher than that of controls. The number of another type of aberrant 5-hydroxytryptamine immunoreactive fibers, which were detected only at 20 weeks of age in the caudate-putamen in LEC rats was significantly higher than that of controls. These results suggest that age-dependent changes in copper concentrations of Long-Evans Cinnamon rats were related to changes in monoaminergic neuron systems.

Improvement of endothelial function and insulin sensitivity with vitamin C in patients with coronary spastic angina: possible role of reactive oxygen species.

OBJECTIVES: This study was designed to examine the effect of antioxidant supplementation on the endothelial function and insulin sensitivity in patients with coronary spastic angina (CSA). BACKGROUND: Insulin resistance may play a key role in coronary heart disease, and there is a possible link between acetylcholine-induced coronary vasoconstriction and hyperinsulinemia in patients with CSA. Endothelial dysfunction is present in the systemic arteries in CSA patients, and reactive oxygen species may cause inactivation of nitric oxide in these patients. METHODS: We measured flow-mediated dilation of the brachial artery using ultrasound technique in 22 patients with CSA and 20 control subjects. We also evaluated glucose tolerance using a 75-g oral glucose tolerance test and insulin sensitivity using steady-state plasma glucose (SSPG) methods in the same patients. RESULTS: The incidence of impaired glucose tolerance was higher in the CSA group than in the control group. Vitamin C infusion augmented flow-mediated dilation and decreased SSPG levels in the CSA group (from 3.27 +/- 0.77% to 7.00 +/- 0.59% [p < 0.001 by analysis of variance (ANOVA)] and from 177.3 +/- 13.3 to 143.1 +/- 14.9 mg/dl [p = 0.047 by ANOVA], respectively) but not in the control group (from 6.47 +/- 0.66% to 6.80 +/- 0.60% and from 119.8 +/- 11.7 mg/dl to 118.1 +/- 11.3 mg/dl, respectively). The steady-state plasma insulin levels were not affected by vitamin C infusion in either group. CONCLUSIONS: Vitamin C improves both endothelial function and insulin sensitivity in patients with CSA. Thus, reactive oxygen species and/or decreased nitric oxide bioactivity may play an important role in the genesis of both endothelial dysfunction and insulin resistance in patients with CSA.

Clear cell chondrosarcoma of the pelvis in a skeletally immature patient.

We report on a case of clear cell chondrosarcoma (CCCS) of the left iliac bone in a 12-year-old skeletally immature boy. Radiographic examination revealed an aggressive osteolytic lesion with areas of mineralization. Fluid-fluid levels were seen on T2-weighted MR images. Laboratory data showed slight elevation of serum alkaline phosphatase. The biopsy specimen showed histological features of CCCS with some resemblance to osteosarcoma, such as prominent irregular osteoid formation among clear tumor cells. Surgical treatment was accomplished without pre- or post-operative chemotherapy. Because of the patient's age, elevated serum alkaline phosphatase, and histopathology with prominent osteoid production, this case could be confused with osteosarcoma. Although CCCS is an extremely rare bone tumor in children, it is important to be aware that it may arise in a skeletally immature patient. CCCS, unlike osteosarcoma, is not treated with neo-adjuvant chemotherapy.

Pax-6 is required for thalamocortical pathway formation in fetal rats.

Pax-6, a transcription regulatory factor, has been demonstrated to play important roles in eye, nose, and brain development by analyzing mice, rats, and humans with a Pax-6 gene mutation. We examined the role of Pax-6 with special attention to the formation of efferent and afferent pathways of the cerebral cortex by using the rat Small eye (rSey2), which has a mutation in the Pax-6 gene. In rSey2/rSey2 fetuses, cortical efferent axons develop with normal trajectory, at least within the cortical anlage, when examined with immunohistochemistry of the neuronal cell adhesion molecule TAG-1 and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling from the cortical surface. A remarkable disorder was found in the trajectory of dorsal thalamic axons by immunostaining of the neurofilament and the neural cell adhesion molecule L1 and DiI labeling from the dorsal thalamus. In normal rat fetuses, dorsal thalamic axons curved laterally in the ventral thalamus without invading a Pax-6-immunoreactive cell cluster in the ventral part of the ventral thalamus. These axons then coursed up to the cortical anlage, passing just dorsal to another Pax-6-immunoreactive cell cluster in the amygdaloid region. In contrast, in rSey2/rSey2 fetuses, dorsal thalamic axons extended downward to converge in the ventrolateral corner of the ventral thalamus and fanned out in the amygdaloid region without reaching the cortical anlage. These results suggest that Pax-6-expressing cell clusters along the thalamocortical pathway (ventral part of the ventral thalamus and amygdala) are responsible for the determination of the axonal pathfinding of the thalamocortical pathway.

Values of the serum components in Japanese black beef steers at farms with high productivity and low frequencies of disease and death in Miyazaki Prefecture.

Few reference values for use in metabolic profile tests for the maintenance of high productivity and the prevention of production diseases have been reported in Japanese Black beef cattle. To obtain basic data, 101 healthy steers at farms with high productivity and low frequencies of disease and death in Miyazaki Prefecture, Japan, were examined for the values of their serum components in this preliminary study. At the later fattening stage (5 to 20 months after introduction), statistically significant increases were observed in the mean serum activities of lactic dehydrogenase, glutamic-oxalacetic transaminase, gamma-glutamyl transpeptidase, and creatine phosphokinase, the mean serum contents of triglyceride, total cholesterol, albumin (Alb), total protein, blood urea nitrogen, magnesium, and vitamin E, and the mean serum calcium (Ca)/inorganic phosphorus (IP) ratio, and statistically significant decreases were seen in the mean serum alkaline phosphatase activity and the mean serum contents of glucose, IP, and vitamin A. The mean serum Alb/globulin ratio and the mean serum Ca and nonesterified fatty acids contents demonstrated no statistically significant changes.

Immunohistochemical localization of neurocan and L1 in the formation of thalamocortical pathway of developing rats.

We used immunohistochemistry to examine possible molecular interactions between the subplate and growing thalamocortical axons in rat fetuses. In the cortical anlage of embryonic day 16 (E16), the subplate first appeared below the cortical plate. Among chondroitin sulfate proteoglycans, phosphacan was uniformly distributed throughout the cortical wall, whereas neurocan was localized only in the subplate at E16. Neural cell adhesion molecules, NCAM-H, TAG-1, and L1, were detected in the cortical anlage. Both cortical neurons and growing axons were diffusely immunopositive for NCAM-H, and TAG-1 immunoreactivity was found on immature neurons and cortical efferent axons but not on thalamocortical axons. L1 immunoreactivity was specifically localized on the growing thalamocortical axons. When the locations of neurocan and L1 were compared in the developing cortex, L1-bearing axons were found to extend to neurocan-immunopositive regions; neurocan immunoreactivity was intense in the subplate at E16, when small numbers of L1-immunoreactive thalamocortical axons began to invade the cortex. At E17, many L1-positive axons were observed in the subplate that expressed neurocan specifically. Double immunostaining showed that L1-positive axons and neurocan immunoreactivity overlapped in the subplate at E17. After E18, neurocan expression gradually extended to the lower part of the cortical plate; it extended to the entire cortex by E21, 1 day before birth. By E21, L1-bearing axons had invaded the lower part of the cortical plate. The present study demonstrated that the neurocan expression precedes growth of L1-bearing thalamocortical afferent fibers. Because neurocan can bind to L1 molecule in vitro, these results suggest that neurocan and L1 play some important roles in pathfinding of the thalamocortical afferent fibers during rat corticogenesis.

Neurons in the caudal ventrolateral medulla projecting to the paraventricular hypothalamic nucleus receive synaptic inputs from the nucleus of the solitary tract: a light and electron microscopic double-labeling study in the rat.

We used light and electron microscopic techniques to investigate the possibility that neurons in the nucleus of the solitary tract (NST) might send projection fibers to neurons in the caudal ventrolateral medulla (CVLM) projecting to the paraventricular hypothalamic nucleus (PVN) by the anterograde and retrograde double labeling method in the rat. The retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, was injected into the PVN, and the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was injected into the NST of each rat. Many retrogradely labeled neurons were encountered in the CVLM, including the A1 region. On the other hand, a dense network of anterogradely labeled terminals was found in the CVLM. Electron microscopic examination revealed synaptic contacts between PHA-L-immunoreactive nerve terminals and dendrites of retrogradely labeled neurons in the CVLM. The results indicate that CVLM neurons projecting to the PVN receive axon terminals of NST neurons.

The POU domain transcription factor Brn-2 is required for the determination of specific neuronal lineages in the hypothalamus of the mouse.

We generated mice carrying a loss-of-function mutation in Brn-2, a gene encoding a nervous system specific POU transcription factor, by gene targeting in embryonic stem cells. In homozygous mutant embryos, migratory precursor cells for neurons of the paraventricular nuclei (PVN) and the supraoptic nuclei (SO) of the hypothalamus die at approximately E12.5. All homozygous mutants suffered mortality within 10 days after birth, possibly because of a complete deficiency of these neurons in the hypothalamus. Although neither developmental nor histological abnormalities were observed in heterozygous mice, the levels of expression of vasopressin and oxytocin in the hypothalamus of these animals were half these of wild-type mice. These results strongly suggest that Brn-2 plays an essential role in the determination and development of the PVN and SO neuronal lineages in the hypothalamus.

Immunohistochemical study of giant cell in glioblastoma.

The clinical and immunohistochemical features of supratentorial (5 patients) and cerebellar (1 patient) glioblastomas, in which giant cells were conspicuous were examined. Three of the patients died within 26 months after the first treatment, and the follow-up period is presently 1 year or less in the remaining patients. The giant cells either showed large and bizarre nuclei or were multinucleated. Both giant and smaller cells excluding neuronal, endothelial and infiltrative cells were positive for GFAP, vimentin, and alpha-1 anti-chymotrypsin. The strong positivity for PCNA staining indicated that the capacity of the giant cells to synthesis DNA was preserved. DNA fragmentation, measured by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine-5'-triphosphate (dUTP)-biotin nick end labeling method, was observed in only 1 patient, who had received radiotherapy just before biopsy, and none of the patients showed bcl-2 positivity. Mutant type of p53 tumor suppressor gene was observed in the giant cells of 3 patients. Giant cell in glioblastoma is of glial origin, synthesizes DNA, and its progression may be related to tumor suppressor gene.

Immobilization of nucleoside on silica gel and specific separation of oligonucleotides.

Deoxyadenosine was immobilized on silica gel, in order to use as HPLC resins for selective separation of oligonucleotides. The longest retention time was observed for the complementary pd(T)4, and increased with decrease of temperature. This fact suggested that the main separation factor was the base pairing between complementary nucleic acid bases. These resins may be useful for separation of components of nucleic acids and polynucleotides as a specific separation system.

Changes in phosphorus-31 magnetic resonance spectra caused by epidural balloon in cats.

Changes in brain high energy phosphate metabolite and pH levels were studied using serial phosphorus (P)-31 magnetic resonance spectroscopy (MRS) in an epidural balloon cat model. The balloon was inflated with 1, 2, 3, or 4 ml of water at 1 ml/sec and deflated after obtaining a spectrum 4 minutes later. Serial spectra, intracranial pressure (ICP), physiological parameters, and the pupil sizes were observed for 1 hour. The mean ICP after balloon inflation increased to between 36 +/- 3 and 130 +/- 6 mmHg. Some animals showed oculomotor paralyses. Phosphocreatine, adenosine triphosphate, phosphodiester, and phosphomonoester levels suddenly decreased after inflation and gradually recovered after balloon deflation. Acidosis progressed with cerebral compression for 4 minutes and gradually recovered. All changes and recovery were volume related. This study demonstrates the potential of P-31 MRS for noninvasive, serial, in vivo measurements of critical high energy phosphate metabolites and intracellular pH after head injury.

[Influence of total body hyperthermia on normal brain tissue].

The influence of total body hyperthermia (TBHT) on normal brain tissue was studied in 40 dogs. The dogs were anesthetized with sodium thiopental (10 mg/kg/hr) intravenously, and were ventilated by artificial respirator. The TBHT was induced by extracorporeal circuit in cooperating a heat exchanger. Rectal temperature was raised to 41.5 degrees C and maintained at 41. 5 -42.0 degrees C for 2 hr (HT period) and was then fallen to normothermia by cooling, Regional cerebral blood flow (CBF) was measured by hydrogen clearance method before heating, during and after TBHT treatment. Brain temperature, rectal temperature, intracranial pressure (ICP), brain tissue pH and electroencephalography (EEG) were monitored continuously during TBHT. Histopathological changes of the brain tissue were studied in dogs killed just after TBHT and 2 weeks after TBHT. Autoregulation of the CBF during HT period was assessed by measuring the regional CBF and the ICP at a state of induced hypo- or hypertension. The brain temperature (at the depth of 5mm under the brain surface) was usually 0.6 degrees C lower than the rectal temperature during HT period. The regional CBF increased from 38.1 +/- 6.5 (mean +/- SD) to 49.1 +/- 9.8ml/100 g/min by raising rectal temperature, and it recovered to a normal value after cooling. The ICP increased from 10.3 +/- 4.2 to 16.8 +/- 3.4 mmHg by raising rectal temperature, and it returned to a normal value after cooling. Brain tissue pH decreased from 7.33 +/- 0.02 to 7.17 +/- 0.09 rapidly when the rectal temperature reached 41.0 degrees C, and then returned to a normal value gradually after the start of cooling.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cyclosporine on the development of cerebral vasospasm in a primate model.

The authors studied the effect of the immunosuppressive drug cyclosporine (CS) on the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) using a primate model of vasospasm. Eighteen monkeys were randomly divided into three groups: a sham-operated control group, an SAH group, and a CS-treated group. To induce SAH, the right side of the circle of Willis was dissected free of the arachnoid and an autologous blood clot was placed around the arteries. In the CS group, CS (5 mg/kg/day) was administered intramuscularly for 7 days after the induction of SAH. The vessel caliber was evaluated on angiograms before the induction of SAH (Day 0) and 7 days after SAH (Day 7). Histological changes and the deposition of IgG in the arterial wall were studied in the three groups. The combined values of the average reduction of the right cerebral arteries at Day 7 was significant (P less than 0.05) in the SAH group (-43.3%) and in the CS group (-31.3%) as compared with the Sham group (-0.7%); however, there was no significant difference between the values in the SAH and the CS groups. In the CS group, the average reduction in vessel caliber of the right middle and anterior cerebral arteries was significantly (P less than 0.05) less than in the SAH group; this did not prove true for the internal carotid artery, however. Although the deposition of IgG in the media and an inflammatory reaction were observed in the spastic arterial wall in both the SAH and CS groups, there was no definitive difference in these immune/inflammatory reactions between the two groups. It is suggested that CS may be helpful in reducing the severity of vasospasm, but may not have a major therapeutic effect, considering its systemic toxicity.

Phosphorus-31 magnetic resonance spectroscopy of cerebral ischemia in cats.

The energy metabolism of the brain was measured in three types of ischemic models in the cat using phosphorus-31 magnetic resonance spectroscopy. The cerebral ischemia was produced as follows. In Group 1, two balloons were inflated in the left subclavian artery and the brachiocephalic trunk. In Group 2, the left middle cerebral artery was occluded through a transorbital approach. A combination of the two was employed in Group 3. Phosphorus-31 magnetic resonance spectra were obtained serially during 2 hours of ischemia. Immediately after occlusion, peaks of phosphocreatine and adenosine triphosphate decreased, whereas the peak of inorganic phosphate increased and split in two. Intracellular pH determined by chemical shift of the inorganic phosphate peak decreased. These changes were more pronounced in Group 3 when compared with the other groups. Histological study showed no infarction in Group 1 and infarcted areas in Groups 2 and 3. The size of the infarcted area in Group 3 was larger than that in Group 2. These results suggest that the model of middle cerebral artery occlusion potentiated with the occlusion of the brachiocephalic trunk and the left subclavian artery by balloon catheters is a reliable stroke model and that phosphorus-31 magnetic resonance spectroscopy is useful to understand the pathophysiological state of cerebral ischemia in vivo.

Development of the neuronal system containing neuropeptide Y in the rat hypothalamus.

In the rat hypothalamus, neuropeptide Y-containing neurons first appeared on day 14.5 of gestation in the arcuate nucleus and in the dorsolateral hypothalamic area. Until birth neuropeptide Y-containing cell bodies increased in number in the arcuate, dorsomedial-lateral and paraventricular nuclei, but disappeared thereafter, but some cells remaining in the arcuate nucleus. In animals treated neonatally with monosodium L-glutamate to destroy the arcuate nucleus, neuropeptide Y-immunoreactivity became evident in many cells scattered in the magnocellular paraventricular and dorsomedial-lateral hypothalamic nuclei on day 16 but not on days 60 and 12. These neuropeptide Y-immunoreactive neurons which appeared in the paraventricular nucleus were also vasopressin-positive. Neuropeptide Y fibers, on the contrary, remarkably diminished in number on day 16, particularly in the paraventricular and dorsomedial-lateral nuclei, and the medial preoptic area, but made a considerable recovery on days 60 and 120. Hence it is probable that, in normal ontogenetic progress, the development of the neuropeptide Y fibers in these areas is inhibitorily affected by that of arcuate neuropeptide Y neurons.

CRF-containing neuron systems in the rat hypothalamus: retrograde tracing and immunohistochemical studies.

By employing a combination of the immunohistochemistry for rat corticotropin-releasing factor (rCRF) and retrograde tracing with biotinylated wheat germ agglutinin (b-WGA) injected into the posterior pituitary (group 1) or into the middle portion of the median eminence (group 2), functionally different populations of CRF neurons were identified in the rat hypothalamus. In the group 1 animals, WGA-labeling was seen not only in the posterior lobe but also in the intermediate lobe, in which CRF fibers exist. In these animals, WGA-labeling occurred for almost all large neurons in the anterior commissural, paraventricular (PV), and supraoptic (SO) nuclei, some of the neurons showing a slight immunoreactivity for anti-rCRF. Conversely, CRF positive neurons appeared in large numbers, some being labeled with WGA, in the caudal periventricular region (CPR), and in the dorsomedial to lateral hypothalamic area (DLH), especially in the latter. In the group 2 animals, WGA was disparsed throughout the subependymal, internal, and external layers of the medial portion of the median eminence, and was taken up by many small cells in the PV, almost half of the cells being immunoreactive for CRF. Slight WGA-labeling further occurred in some large neurons of the PV and SO, and in some cells of the DLH. It is concluded that hypophysiotropic CRF existing in the external layer of the median eminence originates from small neurons located in the PV, whereas CRF distributed in the posterior and intermediate pituitary originates from the magnocellular PV and SO, and from some neurons in the DLH and CPR.

Somatostatin-containing neuron systems in the rat hypothalamus: retrograde tracing and immunohistochemical studies.

By employing a combination of the immunohistochemistry for somatostatin (SRIF) and retrograde tracing with biotinylated wheat germ agglutinin (b-WGA) injected into the posterior pituitary (group 1) or into the median eminence (group 2), functional topography of hypothalamic SRIF neurons was determined in the rat hypothalamus. In group 1, large numbers of WGA-labeled neurons appeared in the rostral periventricular region and in the magnocellular division of the paraventricular and supraoptic nuclei; none of them were SRIF immunoreactive. In group 2, WGA-labeled neurons were numerous in the rostral periventricular region, the parvicellular division of the paraventricular nucleus, and the arcuate nucleus; most of the WGA-labeled neurons in the rostral periventricular region and some in the paraventricular nucleus were SRIF immunoreactive, but none in the arcuate nucleus showed immunoreactivity for SRIF. It is concluded that, in the rat hypothalamus, the locations of neurons containing hypophysiotrophic SRIF are confined within the rostral periventricular region and the parvicellular paraventricular nucleus. Our results do not support previous suggestions that SRIF immunoreactive axons innervate the posterior lobe of the pituitary.

Functional topography of the rat hypothalamic dopamine neuron systems: retrograde tracing and immunohistochemical study.

The origins of the dopamine (DA)-containing nerve terminals in the external layer of the median eminence and in the neurointermediate pituitary were determined in rats by a combination of retrograde labeling with wheat germ agglutinin (WGA) and immunohistochemistry for tyrosine hydroxylase (TH). Biotinylated WGA (b-WGA) was injected into the posterior pituitary (group 1) and into the median eminence (group 2). In group 1 animals, all the magnocellular neurons of the paraventricular, supraoptic, and accessory nuclei, and many parvicellular neurons in the rostral periventricular region (RPR) were labeled with WGA. In group 2 animals, many neurons were labeled in the arcuate nucleus and the RPR, and in a small population of the preoptic-septal region. In group 1 animals, about 39% of TH neurons in the RPR were labeled with WGA, whereas only a few TH neurons (1%) in the arcuate nucleus were labeled with WGA. In group 2 animals, on the contrary, almost all TH neurons (73%) in the arcuate nucleus carried WGA, whereas in the RPR, only some of the TH neurons (19%) were labeled with WGA. It is concluded that DA neurons involved in the hypothalamic-anterior pituitary axis are located in the arcuate nucleus; those involved in neuro-intermediate lobe function in the RPR.