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In this study, we applied a novel psycho-educational horseback riding (PEHR) program to the treatment of four Japanese children with pervasive developmental disorders (PDD) in order to facilitate the acquisition of verbal and nonverbal communication skills. The behavioral changes in each child were evaluated using a psychological and behavioral scale. The scale for evaluating the effect of Human-Equips-Interaction on Mental activity (HEIM scale) was designed to assess the behavioral improvement of children based on the following 10 items: Human relationships, Imitation, Emotional expression, Sudden physical movement, Fixative behavior, Adaptation to change, Visual response, Fear or nervousness, and Verbal and nonverbal communication. After taking part in the PEHR program for several months, all subjects showed remarkably improved HEIM scores and marked improvements were observed in eye contact with others (instructors, side walkers, and leaders) in the riding area. A statistical difference was found in items 1, 2, 3, 6, 7, 8, and 9. However, no statistical difference was found in items 4, 5, and 10. As the program progressed, the children showed enhanced verbal and nonverbal communication skills, and became more expressive in their emotional and empathetic interaction with their parents. These observations suggest that the normal functioning of pleasurable emotions and empathy may facilitate further improvements in joint attention, imitation and empathy, and may result in successful verbal expression by PDD children. Therefore, horseback riding can play a very important role in the psycho-educational support required for the communication ability of PDD children.

The SAMP10 mouse is a model of accelerated ageing in which senescence is characterized by age-related atrophy of the cerebral cortex and limbic structures, poor learning and memory task performance with depressive behaviour and cholinergic and dopaminergic alterations. Here we studied age-related changes in the dendritic arbors and spine density of pyramidal cells in the medial prefrontal cortex of SAMP 10 mice using a quantitative Golgi method. Dendrites of prefrontal neurones gradually retracted with ageing towards the soma with the relative preservation of overall complexity. Apical dendrites were much more severely affected than basal dendrites. The combined length of the apical dendrites and spine density were decreased by 45% and 55%, respectively, in mice at 12 months, compared with mice at 3 months of age. Immunohistochemical and immunoblot analyses indicated that expression of microtubule-associated protein (MAP) 2, a marker of dendrites, decreased in an age-related manner not only in the anterior cortex but also in the posterior cortex and olfactory structures in SAMP10 mice. Decreased expression of MAP2 mRNA caused the decrease in MAP2 protein expression. These results suggest that retraction of apical, but not of basal dendrites, with a loss of spines in prefrontal neurones, appears to be responsible for poor learning and memory performance in aged SAMP 10 mice. It is also suggested that age-related dendritic retraction occurs in a wide area including the entire cerebral cortex and olfactory structures.

Leukotriene B4 is a potent lipid mediator, which has been identified as a potent proinflammatory and immunomodulatory compound. Although there has been robust evidence indicating that leukotriene B4 is synthesized in the normal brain, detailed distribution and its functions in the nervous system have been unclear. To obtain insight into the possible neural function of leukotriene B4, we examined the immunohistochemical distribution of leukotriene A4 hydrolase, an enzyme catalyzing the final and committed step in leukotriene B4 biosynthesis, in the mouse nervous system. Immunoreactivity for leukotriene A4 hydrolase showed widespread distribution with preference to the sensory-associated structures; i.e. neurons in the olfactory epithelium and vomeronasal organ, olfactory glomeruli, possibly amacrine cells, neurons in the ganglion cell layer and three bands in the inner plexiform layer of the retina, axons in the optic nerve and tract up to the superior colliculus, inner and outer hair cells and the spiral ganglion cells in the cochlea, vestibulocochlear nerve bundle, spinal trigeminal tract, and lamina II of the spinal cord. Double immunofluorescence staining demonstrated that most of the leukotriene A4-hydrolase-immunopositive neurons coexpressed calretinin, a calcium-binding protein in neurons. The ubiquitous distribution of leukotriene A4 hydrolase was in sharp contrast with the distribution of leukotriene C4 synthase [Shimada A, Satoh M, Chiba Y, Saitoh Y, Kawamura N, Keino H, Hosokawa M, Shimizu T (2005) Highly selective localization of leukotriene C4 synthase in hypothalamic and extrahypothalamic vasopressin systems of mouse brain. Neuroscience 131:683-689] which was confined to the hypothalamic and extrahypothalamic vasopressinergic neurons. These results suggest that leukotriene B4 may exert some neuromodulatory function mainly in the sensory nervous system, in concert with calretinin.

Chondroitin sulfate proteoglycans have been shown to participate in the pathogenesis of neuronal damages in the injured adult central nervous system (CNS). Upregulated expression of chondroitin sulfate proteoglycans has been reported around the injured sites and depletion of these chondroitin sulfate proteoglycans brings about increased axonal regeneration in the injured adult CNS. To examine if chondroitin sulfate proteoglycans are also involved in the pathologic process of hypoxia-ischemia in the neonatal brain, expressions of three chondroitin sulfate proteoglycans, neurocan, phosphacan, and neuroglycan C, were examined in rat brains after neonatal hypoxia-ischemia. Hypoxic-ischemic rats were produced by ligating the right carotid artery of 7-day-old rats, followed by 8% oxygen exposure. Western blot analysis revealed that in contrast to injured adult CNS, the amount of neurocan was reduced 24 hr after hypoxia in the neonatal hypoxic-ischemic cerebral hemisphere. The amounts of phosphacan and neuroglycan C were also reduced significantly 24 hr after hypoxia at the right injured cortex compared to those at the left cortex. Surprisingly, the immunohistologic staining for phosphacan was conversely intensified both at 24 hr and 8 days after hypoxia at the infarcted area. In addition, the habenula and fascicules retroflexus in the right cerebral hemisphere degenerated and became intensely immunostained with the anti-phosphacan antibody shortly after hypoxia. Hypoxic-ischemic insult may unmask phosphacan epitopes at the injured sites, resulting in intensified immunostaining. Because intensified immunostaining for neurocan and neuroglycan C was not observed, unmasking seems to be specific to phosphacan among these three chondroitin sulfate proteoglycans.

In this report, we investigated whether reactive astrocytes produce neuregulins (glial growth factor 2/heregulin/acetylcholine receptor-inducing activity or neu differentiation factor) and its putative receptors, ErbB2 and ErbB3 tyrosine kinases, in the injured CNSin vivo. Significant immunoreactivities with anti-neuregulin, anti-ErbB2, and anti-ErbB3 antibodies were detected on astrocytes at the injured site 4 d after injury to the adult rat cerebral cortex. To elucidate the mechanisms for the upregulation of neuregulin expression in astrocytes, primary cultured astrocytes were treated with certain reagents, including forskolin, that are known to elevate the intracellular level of cAMP and induce marked morphological changes in astrocytes. Western blot analysis showed that the expression of a 52 kDa membrane-spanning form of a neuregulin protein was enhanced in cultured astrocytes after administration of forskolin. The upregulation of glial fibrillary acidic protein was also observed in astrocytes treated with forskolin. In contrast, inactivation of protein kinase C because of chronic treatment with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate downregulated the expression of the 52 kDa isoform, although other splice variants with apparent molecular sizes of 65 and 60 kDa were upregulated. These results suggest that the enhancement of neuregulin expression at injured sites is induced, at least in part, by elevation in intracellular cAMP levels and/or a protein kinase C signaling pathway. The neuregulin expressed on reactive astrocytes may stimulate their proliferation and support the survival of neurons surrounding cortical brain woundsin vivo.

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan with an epidermal growth factor module that is expressed predominantly in the brain. Cloning studies with mouse NGC cDNA revealed the expression of three distinct isoforms (NGC-I, -II, and -III) in the brain and revealed that the major isoform showed 94. 3% homology with the rat counterpart. The NGC gene comprised six exons, was approximately 17 kilobases in size, and was assigned to mouse chromosome band 9F1 by fluorescence in situ hybridization. Western blot analysis demonstrated that, although NGC in the immature cerebellum existed in a proteoglycan form, most NGC in the mature cerebellum did not bear chondroitin sulfate chain(s), indicating that NGC is a typical part-time proteoglycan. Immunohistochemical studies showed that only the Purkinje cells were immunopositive in the cerebellum. In the immature Purkinje cells, NGC, probably the proteoglycan form, was immunolocalized to the soma and thick dendrites on which the climbing fibers formed synapses, not to the thin branches on which the parallel fibers formed synapses. This finding suggests the involvement of NGC in the differential adhesion and synaptogenesis of the climbing and parallel fibers with the Purkinje cell dendrites.

Crigler-Najjar syndrome (CN) type I, which is characterized by the complete absence of bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT) activity, is inherited as an autosomal recessive trait associated with unconjugated hyperbilirubinemia. Phenobarbital has no effect on the bilirubin concentration in the serum of patients with CN type I. Recently, cDNA for two human liver bilirubin UGT (UGT1A and UGT1D) were isolated, and their genetic organization was determined. The UGT1A (UGT1*1) and UGT1D (UGT1*4) genes each have a unique exon 1, whereas exons 2-5 are common to both genes. It has been predicted that some defect in the exons common to both genes is responsible for the absence of UGT1A and UGT1D activities in CN type I, and five cases with such a mutation have been reported. We describe here a new type of defect in the gene for bilirubin UGT in a patient with CN type I, namely, an abnormality in the exon 1 that is characteristic of the UGT1A gene. This mutation is a single nucleotide substitution, that is, C is changed to A at base position 840 in UGT1A cDNA, and this change results in a stop codon. Our patient is homozygous for the defect, and his nonconsanguineous parents and elder brother, who are clinically normal, are heterozygous for the defective allele. No mutation was detected in any exons of the UGT1D gene. Our results suggest that a homozygous nonsense or deletion mutation is detected not only in the exons common to UGT1A and UGT1D genes but also in unique exon 1 of UGT1A in CN type I.

The protective effect of free radical scavengers against phototoxicity was investigated with tin-protoporphyrin (SnPP)-treated suckling rats. Six kinds of scavengers (L-ascorbic acid, reduced glutathione, α-tocopherol, retinol, uric acid and cystine) were intraperitoneally injected to rats treated with SnPP plus photoirradiation. Among them, L-ascorbic acid was found to be most effective in protecting SnPP-treated rats against phototoxicity. The survival period was markedly prolonged, and the frequency of abnormal behaviors was reduced with the treatment. Lipid peroxidation in vitro with the brain membrane fraction was also suppressed. The other five substances gave only a little antioxidant effect both in vivo and in vitro. The present study shows that L-ascorbic acid may be a promising chemical to prevent the phototoxicity of SnPP.

Bilateral, symmetrical, experimental aneurysms were produced with anastomosed vein flap in the carotid arteries of 24 mongrel dogs. Aneurysms were occluded with latex or silicone balloons on each side and observed angiographically from 2 weeks to 2 months. A histopathological study was performed subsequently using light and scanning electron microscopy. Rupture after balloon embolization occurred in five aneurysms; all of which were incompletely occluded by a silicone balloon. On subsequent angiograms, four silicone balloons and one latex balloon were found to have migrated into the aneurysm, resulting in aneurysmal expansion. Parent artery occlusion was more common with latex balloons than silicone balloons. Histopathologically, residual fresh thrombi, decreased proliferation of fibroblasts within the aneurysmal cavity, and poor endothelialization were present around the silicone balloon. These results suggest that the intra-aneurysmal organization, as seen in the aneurysm occluded by the silicone balloon, will be delayed because the balloon is not fixed within the aneurysm, and that this free-floating and rotating balloon causes repeated trauma to the aneurysm wall, contributing to subsequent enlargement and rupture of the aneurysm. The superior antithrombogenic nature of silicone may be responsible for the bias of such phenomena toward the silicone balloon.

We have demonstrated previously by cDNA vinalysis that VIP is derived together with PHI-like peptide (PHM) from a common precursor (preproVIP) in a humain neuroblastoma cell line called NB-OK-1. The humain neuroblastosma cell line produces and releases the large variants (8K and 18K dalton) of VIP-like immunoreactivity (IR-VIP) in response to dibutyryl cAMP stimulation. The purpose of the present study is to purify the 8Kd vind 18Kd forms of VIP-relaited peptides from the human neuroblastoma culture media and to characterize their structure. IR-VIP in the culture media was fractionated with 40% to 60% of saturated ammonium sulfate (SAS)

In this report we describe our recent histological and biochemical studies to characterize the pathogenic mechanisms of cerebellar hypoplasia exhibited by homozygous Sprague-Dawley-strain Gunn rats and gene mapping of bilirubin UDP-glucuronosyltransferase (BRGT) .Gunn rats are a mutant strain with autosomal recessive trait of hyperbilirubinemia. Homozygous Sprague-Dawley-strain Gunn rats shows marked hyperbilirubinemia and the cerebellar development of the rats is damaged. The severely damaged parts in the cerebellum are known to be anterior and middle lobes while posterior (lobules IXc, IXd and X) are less affected. The most effective day of bilirubin on the cerebellar development is centered on postnatal day 7. It is suggested that the preferential deposition of bilirubin in Purkinje cells of anterior and middle lobes may occur in jaundiced Gunn rat infants, by which the cells are damaged eventually leading to degeneration. The genetic defect in Gunn rats is reported to be deficient in both hepatic BRGT and 3-methylcholanthrene-inducible 4-nitrophenol UDP-glucuronosyltransferase (4-NP-GT) . Both BRGT and 4-NPGT are located on the same chromosome. Sato et al. (1990) isolated a 1763-bp cDNA for rat liver BRGT. The BRGT activity detected by autoradiography on a thin layer chromatogram by transfection of the vector carrying the cDNA into COST cells. The cDNA shares an identical 913-bp sequence with that for rat liver 4-NPGT. A-1 frameshift was found in the locus of 4-NPGT of jaundiced Gunn rats. The result suggests that the multiple defects of UDP-glucuronosyltransferase isoenzymes observed in the Gunn rats may be produced by a single-mutated-locus after an alternative splicing of the identical 913-bp region.

Aims: Neurodegenerative diseases are characterized by ubiquitinated inclusions in selective brain regions. Here we investigated whether the dysfunction of the ubiquitin proteasome system might be involved in the pathogenesis and regional selectivity of neuronal ubiquitinated inclusions using the SAMP10 strain of mouse, an inbred model of age-related cerebral degeneration. Methods: By comparing SAMP10 mice at various ages with SAMR1 and C57BL mice as normal brain ageing controls, we studied morphological features and distribution of inclusions. We measured tissue proteasome activity in different brain regions of mice at various ages by fluorogenic substrate assays. We induced inclusions in cultured neurones by inhibiting the proteasome and analysed changes in the dendritic morphology. Results: Inclusions were formed in association with lipofuscin in neuronal perikarya and occurred most frequently in the limbic-related forebrain structures. There were sparse inclusion-bearing neurones in the non-limbic forebrain. In aged SAMR1 and C57BL, there were far fewer inclusions in the limbic-related forebrain than in aged SAMP10. The proteasome activity in the limbic-related forebrain decreased much more rapidly and remarkably upon ageing (26% activity was detected in 17-month-old compared with 3-month-old mice) in SAMP10 than in SAMR1. The proteasome activity in the non-limbic forebrain did not change significantly with advancing age in either SAMP10 or SAMR1. Proteasomal inhibition enhanced the formation of ubiquitinated inclusions in cultured neurones. Neurones bearing inclusions had shortened neurites. Conclusions: We propose that the regional selectivity of proteasomal impairment is causally related to the selectivity of inclusion formation and associated dendritic degeneration in neurones of ageing SAMP10 mice.

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan with an EGF module. We studied the expression of NGC in the human brain, mainly in the hippocampus, and confirmed some observations by conducting experiments using rat brain. In humans, NGC mRNA was expressed exclusively in the brain, especially in the immature brain. The telencephalon, including the hippocampus and neocortex, showed strong mRNA expression. NGC was immunolocalized to neuropils in the hippocampus and neocortex of the adult rat. RT-PCR experiments showed that four splice variants (NGC-I, -II, -III, and -IV) were expressed in the adult human hippocampus. By Western blotting, the expression as proteins of all splice variants except NGC-II was confirmed in the adult rat hippocampus. NGC-IV, which was first found in the present study, had the shortest cytoplasmic domain among the four variants. NGC-IV mRNA was expressed by neurons, but not by astrocytes, in culture prepared from the fetal rat hippocampus, suggesting that NGC-IV plays a role specific to neurons. In addition, the human NGC gene, which is registered as CSPG5, comprised six exons and was approximately 19 kb in size. In exon 2, a single nucleotide polymorphism resulting in Val188Gly in the NGC ectodomain was observed.

While leukotriene C4 (LTC4) was originally identified as a potent bronchoconstrictor, the compound has versatile biological activities besides inflammatory reactions. Although the high content of LTC4 has been reported in the hypothalamus and median eminence, the precise localization of the compound remained obscure. To elucidate its possible functions in the neuroendocrine systems, we determined immunohistochemical localization of LTC4 synthase, a key enzyme to produce LTC4 using mouse brains. Light microscopy and confocal laser scanning microscopy showed that LTC4 synthase was selectively localized in the vasopressinergic magnocellular neurons of the hypothalamic paraventricular, supraoptic and suprachiasmatic nuclei and in the retrochiasmatic area, as well as in axons that emanated from these neurons to the pars nervosa of the pituitary gland. At subcellular level, however, LTC4 synthase and arginine vasopressin appeared to localize differently within individual neurons. LTC4 synthase immunoreactivity was also observed in the axons of the extrahypothalamic system including the bed nucleus of the stria terminalis, lateral habenular nucleus, midbrain central gray, medial amygdaloid nucleus and ventral septal area, although this immunoreactivity was relatively minor. The other brain regions did not contain LTC4 synthase immunoreactivity. The distribution of LTC4 synthase did not overlap with that of either oxytocin or luteinizing hormone releasing hormone. Therefore, LTC4 is considered to be involved in neural functions of the brain magnocellular vasopressinergic system such as water retention. LTC4 may also be involved in extrahypothalamic vasopressinergic neural functions including the regulation of learning and memory, social recognition memory, sexual and aggressive behavior, etc.

Neurocan is one of the major chondroitin sulfate proteoglycans expressed in nervous tissues. The expression of neurocan is developmentally regulated, and full-length neurocan is detected in juvenile brains but not in adult brains. In the present study, we demonstrated by western blot analysis that full-length neurocan transiently appeared in adult rat hippocampus when it was lesioned by kainate-induced seizures. Immunohistochemical studies showed that neurocan was detected mainly around the CA1 region although the seizure resulted in neuronal cell degeneration in both the CA1 and CA3 regions of the hippocampus. Double-labeling for neurocan mRNA and glial fibrillary acidic protein demonstrated that many reactive astrocytes expressed neurocan mRNA. The re-expression of full-length neurocan was also observed in the surgically injured adult rat brain. In contrast, the expression of other nervous tissue chondroitin sulfate proteoglycans, such as phosphacan and neuroglycan C, was not intensified but rather was either reduced in the kainate-lesioned hippocampus or in the surgically injured cerebral cortex. These observations indicate that induction of neurocan expression by reactive astrocytes is a common phenomenon in the repair process of adult brain injury, and therefore, it can be postulated that juvenile-type neurocan plays some roles in brain repair.

BACKGROUND AND PURPOSE: The pathogenesis and optimal treatment for arterial dissection are still unclear. We devised an experimental model of arterial dissection and observed the morphologic changes with angiography. METHODS: Sixty-four experimental dissections were created in the common carotid arteries of 34 mongrel dogs. After a small incision was made in the arterial adventitia, it was dissected from the media. Elliptical defects (2, 4, 6, and 8 mm in groups I-A, I-B, I-C, and I-D, respectively; n = 47) or longitudinal incisions (4, 6, and 8 mm in groups II-A, II-B, and II-C, respectively; n = 17) were made in the intima distal to the adventitial incision to serve as an entry zone for dissection. RESULTS: Immediately after the lesions were created, the influx of blood into the dissected cavity produced massive subadventitial hematomas, resulting in stenotic changes in all of the arteries, including seven with occlusion. Follow-up (1-week) angiograms demonstrated complete healing, with normal arterial calibers in 11 (79%) of 14 I-A lesions and aneurysm formation in nine (69%) of 13 I-B lesions. All 10 I-D lesions had complete arterial occlusion. Persistent stenosis was observed in all 10 I-C lesions; six of these developed aneurysms. Pathologic examination of the freshly dissected cavities revealed a clot-filled cleft between the media and adventitia. Mature aneurysms, evaluated 3 mo later, had endothelialization within the aneurysmal dome. CONCLUSION: Morphologic changes after arterial dissection are closely related to the size of the intimal entry zone, which may determine whether a dissecting aneurysm forms.

The combination of injection of tin-protoporphyrin IX and photoirradiation produced an acute decrease in serum glucose levels in rats, as well as increased serum lactate dehydrogenase activity, and increased serum levels of creatinine and potassium. A reduction in the serum levels of glucose was particularly noticeable. The severe hypoglycemia may be one of the main causes of death in such rats

The roles of beta-catenin in evagination of the optic primordium in rat embryos were studied using immunostaining. High levels of beta-catenin appeared transiently in the evaginating optic primordium. Evagination of the optic primordium was suppressed in embryos treated with LiCl. In deficient optic vesicles of these embryos, accumulation of beta-catenin was decreased. Deficient optic vesicles also showed suppression of cyclin D1 accumulation and bromodeoxyuridine incorporation, no break in the deposition of laminin and type IV collagen at the basement membrane (BM) and prevention of the change in distribution of microtubules and microfilaments. These results suggest that beta-catenin regulates cell proliferation, breakdown of BM and changes in cell shape in the evaginating optic primordium to cause optic vesicle formation.

The purposes of this study are to determine the molt cycle of the American crayfish, Procambarus clarkii, and to quantify the amounts of the molt-inhibiting hormone (Prc-MIH) in the hemolymph and neurohemal sinus glands during the molt cycle of the American crayfish. The molt cycle was classified into six stages based on the changes in volumes of gastroliths in the stomach and ecdysteroid titers in the hemolymph. A sandwich-type enzyme immunoassay using specific antibodies raised against N-terminal and C-terminal segments of Prc-MIH was developed for the Prc-MIH assay. It is sensitive to as little as 0.5 fmol of Prc-MIH (3.3 x10(-12) M). In the hemolymph, no Prc-MIH could be detected at any of the molt stages tested. However, in the sinus gland, it was demonstrated that the amount of Prc-MIH changes in a molt-stage-specific manner during the molt cycle. It was particularly noteworthy that the initiation of a molting sequence (i.e., entering the early premolt stage) corresponded to the increase in Prc-MIH content in the sinus gland, because the finding is consistent with the hypothesis that crustaceans enter the premolt stage when the MIH secretion from the sinus gland is reduced or ceases.

OBJECTIVE: We developed an in vivo model of growing fusiform aneurysms, using vein grafts to the rat carotid artery. This aneurysm model might demonstrate the pathological features of the development and growth of aneurysms to become giant aneurysms. METHODS: Placement of an interposed femoral vein graft to restore carotid artery flow was performed in Wistar rats. On Day 21, 75% of the grafts (mean diameter, 1.6 mm) were found to be dilated to resemble fusiform aneurysms (mean diameter, 5.82 mm), and 53% of these were giant. Quantitative analysis of the histological findings was performed using image-analyzing software. RESULTS: Histological findings were similar to those for human intracranial giant aneurysms. The average length of the initial grafts in the aneurysm group was 9.1 ± 1.9 mm, and grafts were significantly longer and more tortuous than in the normal graft group (6.4 ± 0.8 mm) (P = 0.01). Cross-sectional areas of the aneurysms (mean, 18.9 mm 2 ) were significantly correlated with the following: 1) the area of intra-aneurysmal thrombosis (mean, 11.1 mm 2 ) (P < 0.0001); 2) the number of intrathrombotic vascular channels (P = 0.005); and 3) the area of dissection, with hemorrhage, between the thrombus and the wall of the aneurysm (mean, 0.72 mm 2 ) (P = 0.0013). Scanning electron microscopic examination showed evidence of endothelial damage associated with growth of the aneurysms. CONCLUSION: Recurrent hemorrhaging from intrathrombotic vascular channels caused dissection between the thrombus and the aneurysm wall, which led to growth of the experimental aneurysms to giant aneurysms. With this model, we demonstrated the growth mechanism of giant fusiform aneurysms.

Cerebella from suckling and adult rats were examined histochemically with 19 different biotin-labeled lectins. Purkinje cells from postnatal rats had a marked ability to combine with many lectins, but minimal ability was found in adult rats except for Con-A, LEL, and MAL lectins. The cell body of Purkinje cells on postnatal day 7 was strongly labeled with 6 lectins (Con-A, LTL, MAL, SJA, UEA-I, and VVA). Only moderate staining was observed with these lectins on postnatal day 5. The dendritic tree of the cells showed a moderate labeling ability with LTL and UEA-I on postnatal days 15 and 20. The dendritic tree was strongly labeled with MAL on postnatal days 10, 15, 20 and adult. Positive reactions were observed in the cells when cerebellar sections from rats on postnatal day 7 were incubated with 3 other lectins (AAL, LEL, and SBA). The cells on postnatal day 7 were rarely labeled with BSL-II, DBA, DSL, LCA, PNA, PSA, RCA120, SSA, STL, and WGA. Purkinje cells on postnatal day 7 may be rich in N-linked oligosaccharides, with terminal sugar structures that resemble blood-group-related antigens (type H) and/or tumor-related antigens. These glycoconjugates may be present at low levels in the Purkinje cells of adult rats. Dendrites of Purkinje cells of adult rats were strongly labeled by Con-A, LEL, and MAL. The dendrites of Purkinje cells may be rich in highly branched oligosaccharides.

Gilbert's and Crigler-Najjar syndromes are characterised by unconjugated hyperbilirubinaemia due to complete and partial absence of bilirubin UDP-glucuronosyltransferase (UGT). Nucleotide sequences of the genes for bilirubin UGT were analysed in six patients with Gilbert's syndrome. All patients had a missense mutation caused by a single nucleotide substitution and the mutations were heterozygous. In addition, relatives of patients with Crigler-Najjar syndrome types I and II, and of those with Gilbert's syndrome were analysed. All ten relatives with mild hyperbilirubinaemia were heterozygotes with respect to each defective allele. These results suggest that Gilbert's syndrome is inherited as a dominant trait.

We examined the effects of cobalt-mesoporphyrin (CoMP) in vitro. The porphyrin inhibited the activity of rat splenic heme oxygenase but scarcely stimulated peroxidation of lipids in a membrane fraction from rat brain during photoirradiation. The apparent inhibition constant for CoMP was 344 nM. It is suggested that CoMP may be a promising candidate for a chemopreventive of neonatal hyperbilirubinemia that is not associated with phototoxicity.

Lipid peroxidation induced by Sn-protoporphyrin (SnPP) plus photoirradiation was investigated in vivo and in vitro using nonjaundiced Gunn rats. Membrane lipids from young adult rat brain were peroxidized by SnPP plus photoirradiation depending on the SnPP concentration and photoirradiance. Similarly, coadministration of SnPP and photoirradiation to suckling rats increased lipid peroxides in the whole blood and was found lethal. The influence of the wavelength distribution of light sources was also examined by using blue-white and green fluorescent lights. The photodynamic effect by green light irradiation whose energy distribution had no overlap with the Soret band of SnPP was about half of that produced by blue-white light with regard to the membrane peroxidation and the lethal effect on neonatal rats. We therefore conclude that the combination of SnPP and photoirradiation is a potentially hazardous treatment of neonatal jaundice.

Activities of acid phosphatase and beta-glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although beta-glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.

Developing homozygous (jj) and heterozygous (j+) Gunn rat cerebella were examined histologically from postnatal days 5 to 60. Sagittal sections of the cerebellar vermis of jj rats revealed that the anterior and medial lobes were significantly smaller in area than in j+ rats on and after postnatal day 10. However, the nodulus did not display significant differences in jj rats. Two classes of acid phosphatase (ACPase)-positive cells (L cells and S cells), and lipid granule-containing S cells were recognized exclusively in the jj rat cerebellum during the postnatal period studied. S cells, which are probably microglia, had an oval dark nucleus and contained many lysosomes, some of which contained lipid droplets. They appeared in all the lobules except the nodulus on day 5 and reached maximum in incidence by day 15. They were distributed all over the cerebellar layers including the white matter. Lipid granule-containing S cells appeared on postnatal day 10 and were most abundant in severely affected lobules, such as the declive and tuber, on day 30. Purkinje cells of jj rats showed vacuolation in their cytoplasm on and after postnatal day 5. After 20 days of life, the number of Purkinje cells in anterior- and medius-lobus were markedly decreased. Some severely damaged Purkinje cells became L cells with an extremely high ACPase activity. They appeared initially on postnatal day 15 and increased in number until day 30. No L cells were observed in the nodulus. These show that the severely damaged Purkinje cells and ACPase-positive and lipid granule-containing microglia cells are most abundant in the late- and intermediate-maturing regions of the vermis. Since they are either rare or absent in any earlier-maturing region, the nodulus, these data suggest toxic effects of bilirubin in the cerebellum are closely related to the ontogenic development of the individual cerebellar lobules.

Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.

Postnatal changes in cerebellar wet weight and the effect of phototherapy were studied using a new strain of Gunn rats with a genetic background of the Sprague-Dawley strain (SDGR). The cerebellar wet weight of homozygous rats reached a maximum at postnatal day 10 and then it gradually decreased up to day 20, suggesting that the cerebellar hypoplasia of the rats was a consequence both of cellular underdevelopment and degeneration. Histological examinations of the 10 cerebellar lobules showed that lobules I-VIII of homozygotes were severely damaged while lobules IX and X were less affected, coinciding with previous reports of Wistar strain Gunn rats. Photoirradiation with blue light of homozygous rats from day 1 through 15 markedly improved their cerebellar development, and the cerebellar wet weight of these rats was almost comparable to that of heterozygous rats as examined at day 30. Photoirradiation from day 1 to 5 protected, though not fully, lobules I-V and VIII against damage, but not lobules VI and VII at all. Photoirradiation from day 6 to 10 prevented all the lobules from hypoplasia. Irradiation from day 11 to 15 was effective only at the tips of lobules VI and VII. These results indicate that the cerebellar hypoplasia of jaundiced Gunn rats can be effectively prevented by a series of photoirradiation during a particular period after birth and also that the most effective period is different among the lobules.

Top of pageAbstract ABSTRACT: Our study was undertaken to examine the pharmacological and biological effects of tin-protopor-phyrin, a competitive inhibitor of heme oxygenase, on 5-or 6-day-old homozygous (j/j) Gunn rats with hereditary unconjugated hyperbilirubinemia. When j/j neonates were injected subcutaneously with 20 μmol of tin-protopor-phyrin/kg of body weight, hepatic heme oxygenase activity decreased to 30% of the initial level 2 h after administration and remained low during the next 46 h. However, the reduction of serum bilirubin was more rapid and transient, reaching the minimum value (40% of the initial level) at 1 h and increasing thereafter at a rate almost comparable to that in nontreated j/j rats. The mortality rate of j/j rats was strikingly reduced by the administration of 1 to 100 μmol of tin-protoporphyrin/kg; the most effective dose was 5 μmol/kg (8% compared with 80% in non-treated j/j rats). However, the protective effect of tin-protoporphyrin on bilirubin cerebellopathy (cerebellar hypoplasia) was less marked than expected. Possible implications of our results are discussed.

Calmodulin is a small, acidic calcium-binding protein concentrated in the nervous system. The immunohistochemical localization of calmodulin in the developing mouse brain was studied light-microscopically using the specific anti-calmodulin IgG which was elicited in the rabbit with native calmodulin of bovine brain. The immunoreactivity of the cell somata and their processes found in the adult brain did not appear simultaneously but in some sequential order with the regions of the brain. On the other hand, immunoreactivity disappeared during the development. This reaction of disappearance was also abolished by the neutralization of the antibody with calmodulin. From the results of the appearance as well as the disappearance of the immunoreactivity, the pattern of the immunostaining reached that of the adult by 17 days of age.

The surface change of the egg of the teleost, Oryzias latipes, during fertilization was observed with a scanning electron microscope. The microvilli of the outer surface of the unfertilized egg show a slight difference in density between the animal and vegetal pole areas. In the initial step of the breakdown of cortical alveoli (CA), several small holes or gapes are formed at the apical part of the CA membrane, becoming a large aperture from which the alveolar contents are discharged. The formation of microvilli is observed on the inner surface of the exposed cavity left by the CA, starting from the periphery of the aperture and propagating throughout the whole inner surface in accompaniment with the release of the alveolar contents. After the completion of CA breakdown, the CA membrane cannot be distinguished from the original egg plasma membrane.

Homozygous (j/j) and heterozygous (j/+) newborn Gunn rats of the Sprague-Dawley strain were photoirradiated for 24 h at scheduled postnatal days and the effects of irradiation on the cerebellar development were examined at 30 days of life. Improvement of the survival rate was the most notable effect of photoirradiation. A single 24-h dose of photoirradiation during a period of postnatal days 4-11 effectively prevented hypoplasia in the j/j rat cerebellum. No prevention by light was observed at days 3 and 12. It was found that the most effective day of irradiation on the cerebellar development of j/j rats was centered on postnatal day 7. When plasma bilirubin was assessed during the period of postnatal days 7-10, a distinct diminution of the concentration was observed, restricted to only the period of the light treatment. Although there were some differences in the effective day as well as in the degree of efficacy of phototherapy among cerebellar lobules or sublobules, day 7 was the most critical for cerebellar hypoplasia due to bilirubin.

Cerebella of 20-day-old homozygous (jj) and heterozygous (j+) Gunn rats were examined histochemically for the presence of lipids. The j+ rat cerebellum showed no lipid granules, whereas many granules were recognized in the hypoplastic jj rat cerebellum. The degree of appearance of lipid granules differed considerably among the lobes – a high content in the declive and tuber but none in the nodulus. Two kinds of cells showed a strong acid phosphatase activity and contained many lipid granules. It is suggested that the accumulation of lipids, probably cholesteryl esters, is closely related to the enhanced activity of the lysosomal system in the hypoplastic jj rat cerebellum severely injured by bilirubin.