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The ascidian Styela clavais an ecologically important species that is distributed along coastal regions worldwide. It has a long history as a model animal for evolutionary and developmental biology research owing to its phylogenetic position between vertebrates and invertebrates, and its classical mosaic expression patterns. However, the standard developmental atlas and protocols and tools for molecular manipulation of this organism are inadequate. In this study, we established a standard developmental table and provided a web-based digital image resource for S. clavaembryogenesis at each developmental stage from fertilized eggs to hatching larvae by utilizing confocal laser microscopy and 3D reconstruction images. It takes around 10 h for fertilized eggs to develop into swimming larvae and 20–30 min to complete the tail regression processes at the metamorphic stage. We observed that the notochord cells in S. clavaembryos did not produce an extracellular lumen like Ciona robusta, but showed polarized elongation behaviors, providing us an ideal comparative model to study tissue morphogenesis. In addition, we established a chemical-washing procedure to remove the chorion easily from the fertilized eggs. Based on the dechorionation technique, we further realized transgenic manipulation by electroporation and successfully applied tissue-specific fluorescent labeling in S. clavaembryos. Our work provides a standard imaging atlas and powerful genetic tools for investigating embryogenesis and evolution using S. clavaas a model organism.

NMDA glutamate receptors have key roles in brain development, function and dysfunction. Regulatory roles of D-serine in NMDA receptor-mediated synaptic plasticity have been reported. Nonetheless, it is unclear whether and how neonatal deficits in NMDA-receptor-mediated neurotransmission affect adult brain functions and behavior. Likewise, the role of D-serine during development remains elusive. Here we report behavioral and electrophysiological deficits associated with the frontal cortex in Pick1knockout mice, which show D-serine deficits in a neonatal- and forebrain-specific manner. The pathological manifestations observed in adult Pick1mice are rescued by transient neonatal supplementation of D-serine, but not by a similar treatment in adulthood. These results indicate a role for D-serine in neurodevelopment and provide novel insights on how we interpret data of psychiatric genetics, indicating the involvement of genes associated with D-serine synthesis and degradation, as well as how we consider animal models with neonatal application of NMDA receptor antagonists.

A case of transient hyperphenylalaninaemia with a maturational delay of dihydropteridine synthesis is described. With the Guthrie test, the patient showed a blood phenylalanine level of 38 mg dl-1, which had fallen to a normal value without a phenylalanine restricted diet by 3 months of age. The neopterin level and the neopterin to biopterin ratio in the patient's urine were very high at 19 days of age. The blood phenylalanine level did not decrease when tetrahydrobiopterin (2.5 mg kg-1) was administered at 19 days of age, while administration of tetrahydrobiopterin (7.5 mg kg-1) at 20 days of age had decreased the blood phenylalanine level to 50% of the preloading level after 24 h. The oral phenylalanine loading test showed the pattern of classic phenylketonuria (PKU) at 15 days of age, but it showed the normal pattern at 1 year 8 months of age.

A case of transient hyperphenylalaninaemia with a maturational delay of dihydropteridine synthesis is described. With the Guthrie test, the patient showed a blood phenylalanine level of 38 mg dl-1, which had fallen to a normal value without a phenylalanine restricted diet by 3 months of age. The neopterin level and the neopterin to biopterin ratio in the patient's urine were very high at 19 days of age. The blood phenylalanine level did not decrease when tetrahydrobiopterin (2.5 mg kg-1) was administered at 19 days of age, while administration of tetrahydrobiopterin (7.5 mg kg-1) at 20 days of age had decreased the blood phenylalanine level to 50% of the preloading level after 24 h. The oral phenylalanine loading test showed the pattern of classic phenylketonuria (PKU) at 15 days of age, but it showed the normal pattern at 1 year 8 months of age.

The role of the renin-angiotensin system in blood pressure control and in the development of hypertension was investigated by generating transgenic mice carrying the rat renin or angiotensinogen gene or both genes under the control of the mouse metallothionein I promoter. The systolic blood pressure was significantly elevated in transgenic mice carrying both transgenes but was maintained normally in those bearing either of the transgenes. The transgene was effectively and properly transcribed to form the mature mRNA in the transgenic mice. The production of rat renin and angiotensinogen in the transgenic mice carrying the corresponding transgene was also verified by immunoanalyses of these proteins. Furthermore, the specific angiotensin-converting enzyme inhibitor captopril was effective in reducing the elevated blood pressure of the hypertensive transgenic mice. These results indicate that the combined action of the exogenous rat renin and angiotensinogen is responsible and necessary for elevation of blood pressure in the hypertensive transgenic mice.

Fatty acids of plasma and erythrocyte membrane sphingomyelin were determined by gas chromatography-mass spectrometry in adrenoleukodystrophy (ALD) without adrenal insufficiency. Mass chromatogram tracing with the ion at m/z 143 [(CH2)6 COOH3]+ showed increases of saturated very long chain fatty acids in plasma and erythrocyte membrane sphingomyelin in ALD. The C26:0/C22:0 ratios in plasma were 0.121, 0.057 and 0.007 in cases 1 and 2, and a control subject, respectively. The C26:0/C22:0 ratios in erythrocyte membrane sphingomyelin were 0.386, 0.211 and 0.093 in cases 1 and 2 and the control subject, respectively. The demyelinating process of ALD was clearly observed in both the inversion recovery 2100/500 and spin echo 2100/80 scans on magnetic resonance imaging. The magnetic resonance image in case 1 revealed widespread demyelinated lesions, involving almost the entire cerebrum and cerebellum, at 4 years after the onset, while that in case 2 revealed demyelinated lesions mainly limited to parieto-occipital areas at 1 year after the onset.

We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.

Transforming growth factors beta (TGF-beta s) and activins induce and inhibins block secretion of follicle-stimulating hormone by rat GH3 pituitary tumor cells. Cheifetz et al. (Cheifetz, S., Ling, N., Guillemin, R., and Massagué, J. (1988) J. Biol. Chem. 263, 17225-17228) reported that GH3 cells express a approximately 50-kDa surface protein, termed the type IV TGF-beta receptor, that directly binds all of these peptide hormones. Here we show that GH3 cells express the previously identified type I and type II receptors for TGF-beta and activin-A. Immunoprecipitation of affinity-labeled surface binding proteins with antisera specific to known receptors demonstrated independent heteromeric complexes of TGF-beta types I and II receptors and of activin types I and II receptors. As judged by ligand-binding and cross-linking analysis, TGF-beta binding to the TGF-beta receptors is not inhibited by activin-A and activin-A binding to its receptors is not inhibited by TGF-beta. Screening of a cDNA library from GH3 cells for potential receptor serine-threonine kinases yielded the known types I and II TGF-beta and activin receptors. The presumed common intracellular signaling pathway for TGF-beta and activin in GH3 cells appears to be mediated by distinct cell-surface receptors.

βγ Subunits of heterotrimeric GTP-binding proteins (Gβγ) activate the inwardly rectifying muscarinic K+channel, GIRK1. The significant role for the carboxyl (C) terminus of GIRK1 in this interaction has been suggested. However, it is still unknown whether Gβγdirectly interacts with GIRK1. To elucidate the molecular basis of Gβγ-activation of GIRK1, we examined the binding properties of Gβγto the C terminus of GIRK1 cloned from mouse brain cDNA library (MB-GIRK1). The C terminus of MB-GIRK1 fused with glutathione S-transferase directly bound to purified Gβγ. Incubation of the C terminus with Gipretreated with GTPγS, but not with GDP, resulted in the binding of Giβγto the protein. Purified Gα-GDP, but not Gα-GTPγS, inhibited the binding of Gβγto the fusion protein. These results indicate that Gβγdissociated from Gαmay directly bind to the C terminus of GIRK1.

Slowly activating IsK channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative treatment reduced the resulting current IsK, while no inhibition was observed for IsK protein mutants carrying a Ser mutation instead of a highly conserved Cys residue in the intracellular domain. In contrast, Hg2+, which may not only oxidize thiol groups but also form chelates with dibasic amino acids, caused a use-dependent, positive regulation of IsK. This effect was reversed in an IsK protein mutant with a deletion in the extracellular domain. These data suggest opposite effects of peroxides and Hg2+ on IsK, a peroxide-mediated IsK inhibition by intracellular oxidation and a Hg(2+)-mediated IsK increase, caused by extracellular Hg2+ chelation of the IsK protein.

Gene deletions were screened in 49 Japanese Duchenne muscular dystrophy patients from 43 families, using the polymerase chain reaction. Enzymatic amplification was carried out on six regions prone to deletion. Fifteen of 43 families (33%) had gene deletions in at least one of the six regions. This frequency was almost the same as that previously reported in Caucasians. The mid-part of the dystrophin gene was the location most frequently deleted. The frequency of deletion of the region encompassing exon 45 was higher in Japanese families (18.4%) than in Caucasians.