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Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle. Although a combination of molecular methods have been used including DNA sequencing of all 10 exons and exon-intron boundaries of OTC gene, only approximately 80% of patients with OTC deficiency are found to have mutations. We report two known and three novel mutations of the OTC gene in five Japanese patients including two neonatal-onset, one late-onset, and two symptomatic female patients. Known nonsense mutations (c.578G>A and c.421C>T) were detected in a neonatal-onset male and a symptomatic female patient, respectively. Mutation analysis revealed two novel mutations including one splice site mutation (c.386+1G>C) in a symptomatic female patient and one missense mutation (c.515T>A) in a late-onset male patient. In the remaining case, which was a neonatal-onset male patient, no mutation was disclosed by direct sequencing of all 10 exons and their flanking intron sequences. Therefore, OTC mRNA in the liver was analyzed by RT-PCR, and remarkably, a 135-nt insertion was detected between exons 5 and 6. Genomic DNA analysis of intron sequences revealed a single nucleotide change at 265 bp downstream from the 3' end of exon 5, which created the novel splice acceptor site. Thereby, a 135-nt exon was created from the central part of an intron sequence. This is the first report of mutation deep in the intronic sequence in the OTC gene. Molecular analysis using genomic DNA and mRNA will increase the mutation detection ratio in the OTC gene.

Sotos syndrome is caused by the haploinsufficiency of the NSD1 gene located in 5q35. More than 70% of the Japanese cases carry microdeletions encompassing of this gene, while point mutations are common in Caucasians. Only 15 familial cases of Sotos syndrome have been reported and all cases shown to have not microdeletions but point mutations. We identified the first Japanese familial case (mother and 3 children). They carry the same mutation at splice donor site of intron 13 (IVS13+1G>A), which results in the in-frame skipping of exon 13. This is also the first familial case caused by the mutation of the splice donor site. Each member of this family showed variable phenotypes and mental development. The present report will contribute to further understanding of genotype-phenotype correlation in Sotos syndrome.

Hypoxia-ischemia (HI) causes injury to oligodendrocytes (OLs), cells which create the myelin sheath in the developing brain. OLs pass successively through progenitor and immature stages during differentiation into mature OLs. Only the OLs in the progenitors stage can express the platelet-derived growth factor-a receptor (PDGF-R(alpha)) so that its expression is one of the cellular markers of OL progenitors. Activation of PDGF-R(alpha) results in OL proliferation, but not OL differentiation. To study the response of OL progenitors after neonatal HI brain injury, we investigated the expression of PDGF-R(alpha) in a neonatal rat stroke model (combination of left common carotid artery ligation and exposure to 8% O2 for 2 h). In the injured cerebral cortex, PDGF-R(alpha) mRNA levels increased significantly (p<0.01) with a peak at 0.5 h after HI insult, and returned to baseline levels within 48 h post-injury. PDGF-R(alpha) protein levels increased significantly at 72-96 h (p<0.05) and then returned to basal levels. Immunohistochemistry showed clear staining of PDGF-R(alpha) only in the injured cerebral cortex at 72 h after HI insult. In contrast, no staining was observed in the cortex of sham-operated controls. These results indicate that the expression of PDGF-R(alpha) increases rapidly and transiently only in the injured cerebral cortex after HI insult and may play a protective role through modulating the glial differentiation under the condition of cellular damage in the developing brain.

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders characterized by degeneration of anterior horn cells in the spinal cord, and leads to progressive muscular weakness and atrophy. At least three SMA-related genes have been identified: SMN1, NAIP and p44t. We analyzed these genes in 32 SMA patients and found that the SMN1 gene was deleted in 30 of 32 patients (94 %), irrespective of clinical type. The NAIP gene was deleted in 6 patients and its deletion rate was higher in type I patients than that in type U or V. Further, in type I patients lacking the NAIP gene, deterioration in their respiratory function is more rapid than in those type I patients retaining the NAIP gene. Since complete p44t deletion was observed in only 3 patients, the correlation between the p44t deletion and severity of SMA remained ambiguous. We concluded that the NAIP deletion was closely related to the clinical severity of SMA and was a predictive marker of SMA prognosis, while the SMN1 deletion did not correlate with clinical severity.

Heat shock proteins (HSPs) are immediately expressed in neuronal and glial cells under various stressful conditions and play a protective role through molecular chaperones. We investigated the characteristics of the induction manner of heme oxygenase-1 (HO-1) and HSP70 in rat C6 glioblastoma cells. In heat treatment (42 degrees C for 30 min), C6 cells expressed high level of HO-1 and HSP70 mRNAs within 30-60 min, and their proteins at 3 hrs. Heat-induced expressions of HSPs mRNAs were completely inhibited with actinomycin D, suggesting the transcriptional regulation. Oxygen-glucose deprivation (OGD), cystine-free (inhibition of synthesis of glutathione), cyto-toxic (ethanol, sodium butyrate) treatments resulted in different expression manners between HO-1 and HSP70, which suggested that HO-1 and HSP70 play different protective roles against a variety kind of stressful conditions in glial cells. C6 cells can differentiate toward both astrocyte and oligodendrocyte directions. Treatment with dibutyryl cyclic AMP (cAMP) induces expression of glial fibrillary acidic protein (GFAP), a marker of astrocytes, and treatment with retinoic acid (RA) induces expression of myelin proteolipid protein (PLP), a marker of oligodendrocytes, respectively. Heat treatment before the initiation of differentiation by RA reduced the RA-induced expression of PLP mRNA profoundly, but not in GFAP mRNA level induced by cAMP. Heat treatment after the initiation of differentiation by cAMP or RA accelerated the expression of GFAP or PLP mRNAs. Astroglial differentiation by cAMP reduced the heat-induced expressions of HSPs mRNAs, but no change with RA pre-treatment. These results suggested that HSPs may modulate the glial differentiation in the developing brain. On the contrary, glial differentiation may give influence on the stress-induced HSPs expression. The timing of stressful damages, resulting in the expression of HSPs, on the developing brain is critically important for the pathogenesis of glial lesion. In the heat-treated C6 cells, the expression of platelet-derived growth factor (PDGF) receptor-alpha mRNA was significantly decreased. HSPs may have ability to induce the glial differentiation in part through down-regulation of the PDGF pathway.

Background: It is essential to evaluate the renal function for the management of high risk neonates. Color Doppler ultrasound technique can provide a useful information to evaluate the neonatal renal artery blood flow velocities. This study was performed to obtain the normative data of renal blood flow velocities in preterm and fullterm neonates and to compare the renal blood flow velocities with the aortic blood flow velocities. The normal volumetric state of fluid balance and renal function are essential in the management of the sick neonates., Methods: The renal peak systolic blood flow velocity (renal PSFV), the renal mean blood flow velocity (renal MFV), the renal end diastolic blood flow velocity (renal EDFV) and the renal resistance index (renal RI) and also the cardiac output, the aortic peak systolic blood flow velocity (aortic PSFV) and the aortic mean blood flow velocity (aortic MFV) were serially recorded from one to 7 days after birth in 16 preterm and one to 5 days in 23 normal fullterm neonates., Results: The renal PSFV was significantly increased with the postnatal age, and the renal MFV was significantly increased between three and five days of age in the preterm neonates. In the fullterm neonates there was no significant change of the renal PSFV between one and five days of age, but the renal MFV was significantly increased between three and five days of age. There were no statistically significant differences of the renal PSFV, the renal MFV, the renal EDFV and the renal RI between the preterm and the fullterm neonates in the first five days after birth. The cardiac output in the preterm neonates was higher than that in the fullterm neonates: on day 3; 313 +/- 59 vs. 254 +/- 40 ml/kg/min. (p < 0.001) and on day 5; 357 +/- 95 vs. 280 +/- 35 ml/kg/min. (p < 0.01). The renal blood flow velocities were not significantly correlated with the aortic blood flow velocities in the normal fullterm neonates, whereas, in the preterm neonates the renal PSFV correlated with the cardiac output (r = 0.35, p < 0.01), the aortic PSFV (r = 0.45, p < 0.001) and the aortic MFV (r = 0.39, p < 0.01), and the renal MFV also correlated with the aortic PSFV (r = 0.30, p < 0.05) and the aortic MFV (r = 0.32, p < 0.05)., Conclusions: The increased renal flow velocities with the postnatal age in the preterm infants might depend on the increased cardiac output.

Accumulating evidence has demonstrated that protein kinase C (PKC) and protease nexin-1 (PN-1) may be involved in neuronal differentiation including migration, neurite outgrowth, target recognition, and synaptogenesis. We investigated the potential roles of PKC and PN-1 in neurite outgrowth of human neuroblastoma cell line, GOTO. Upon withdrawal of serum GOTO cells extended neurite processes within 3 h and formed fine network of neurites after 24 h. This morphological change was completely inhibited by thrombin and phorbol-12-myristate-13-acetate (PMA). Withdrawal of serum increased the neurofilament (NF)-L and -M mRNA levels and thrombin did not inhibit the effect of withdrawal of serum. A potent PKC inhibitor, H-7 induced neurite outgrowth in the presence of serum, however, it did not increase the NF mRNA levels. Actinomycin D and cycloheximide did not inhibit the initial neurite outgrowth induced by withdrawal of serum, while these inhibited the increase in the NF mRNA levels. Thrombin retracted the serum depletion-induced neurites but did not retract the neurites induced by H-7. The specific activity and subcellular localization of PKC did not differ between GOTO cells cultured in serum-containing and -free media for 12 h. The serine protease inhibitory activity was undetectable in the serum-free conditioned medium of GOTO cells but the PN-1 mRNA was clearly detected by Northern blot analysis to a less extent than glial cells. Withdrawal of serum or treatment with H-7 did not increase the PN-1 mRNA level in GOTO cells, but thrombin increased its level about 7 folds in serum-free condition. These results indicate that the initial neurite outgrowth requires neither new RNA nor protein synthesis, and that PKC negatively regulates neurite outgrowth and thrombin blocks neurite outgrowth through PKC-dependent pathways.