EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

In mouse cerebral corticogenesis, neurons are generated from radial glial cells (RGCs) or from their immediate progeny, intermediate neuronal precursors (INPs). The balance between self-renewal of these neuronal precursors and specification of cell fate is critical for proper cortical development, but the signaling mechanisms that regulate this progression are poorly understood. EphA4, a member of the receptor tyrosine kinase superfamily, is expressed in RGCs during embryogenesis. To illuminate the function of EphA4 in RGC cell fate determination during early corticogenesis, we deleted Epha4 in cortical cells at E11.5 or E13.5. Loss of EphA4 at both stages led to precocious in vivo RGC differentiation toward neurogenesis. Cortical cells isolated at E14.5 and E15.5 from both deletion mutants showed reduced capacity for neurosphere formation with greater differentiation toward neurons. They also exhibited lower phosphorylation of ERK and FRS2α in the presence of FGF. The size of the cerebral cortex at P0 was smaller than that of controls when Epha4 was deleted at E11.5 but not when it was deleted at E13.5, although the cortical layers were formed normally in both mutants. The number of PAX6-positive RGCs decreased at later developmental stages only in the E11.5 Epha4 deletion mutant. These results suggest that EphA4, in cooperation with an FGF signal, contributes to the maintenance of RGC self-renewal and repression of RGC differentiation through the neuronal lineage. This function of EphA4 is especially critical and uncompensated in early stages of corticogenesis, and thus deletion at E11.5 reduces the size of the neonatal cortex.     

The scyphomedusan fauna of the Japan Trench: preliminary results from a remotely-operated vehicle

A series of dives was conducted during April/May 2002 in a variety of water masses over the Japan Trench using the remotely-operated vehicle ROV HyperDolphin. The scyphomedusan fauna was catalogued, resulting in the first records of Atolla vanhoeffeni and Atolla russelli from Japanese waters. A new genus and species referable to the family Paraphyllinidae was also recognized. The ulmarid scyphomedusa Poralia rufescens is by far the most common scyphomedusa in these waters. Its distribution was determined by water mass structure and it was not present in subducted Oyashio Current-derived waters occurring within its usual depth range. Temperature and salinity affected its distribution more than dissolved oxygen concentrations. Distributional information, data on the physico-chemical parameters of the water column, and notes on biological associations with scyphomedusae are introduced.     

Loss of Borealin/DasraB leads to defective cell proliferation, p53 accumulation and early embryonic lethality

Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.     

Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558

A succinate-coenzyme Q reductase (complex II) was isolated in highly purified form from Ascaris muscle mitochondria by detergent solubilization, ammonium sulfate fractionation and gel filtration on a Sephadex G-200 column. The enzyme preparation catalyzes electron transfer from succinate to coenzyme Q1 with a specific activity of 1.2 mumol coenzyme Q1 reduced per min per mg protein at 25 degrees C. The isolated complex II is essentially free of NADH-ferricyanide reductase, reduced CoQ2-cytochrome c reductase and cytochrome c oxidase and consists of four major polypeptides with apparent molecular weights of 66 000, 27 000, 12 000 and 11 000 and two minor ones with Mr of 36 000 and 16 000. The complex II contained cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria, at a concentration of 3.6 nmol per mg protein, but neither other cytochromes nor quinone. The cytochrome b-558 in the complex II was reduced with succinate. In the presence of Ascaris NADH-cytochrome c reductase (complex I-III) (Takamiya, S., Furushima, R. and Oya, H. (1984) Mol. Biochem. Parasitol. 13, 121-134), the cytochrome b-558 in complex II was also reduced with NADH and reoxidized with fumarate. These results suggest the cytochrome b-558 to function as an electron carrier between NADH dehydrogenase and succinate dehydrogenase in the Ascaris NADH-fumarate reductase system.     

ESR studies on iron-sulfur clusters of complex II in Ascaris suum mitochondria which exhibits strong fumarate reductase activity

Complex II of Ascaris suum mitochondria, which functions as fumarate reductase in physiological conditions, contains three types of iron-sulfur clusters. These correspond to clusters S-1, S-2 and S-3 and are distinguishable by low-temperature ESR studies. Cluster S-1 is reduced by succinate, giving ESR signals with gz, gy and gx values at 2.033, 1.939 and 1.920. The existence of cluster S-2 is suggested by an enhancement of the S-1 spin relaxation induced upon reduction of S-2 by dithionite. Cluster S-3 is ESR detectable under air-oxidized conditions and gives a strong signal at g = 2.025. Cluster S-3 was only partially reduced even with an excess amount of sodium succinate, which is a common characteristic of fumarate reductase but this is not seen in the mitochondrial complex II.     

Production of 4-hydroxyfuranones in simple media by fermentation

Both 2,5-dimethyl-3(2H)-furanone (DMHF) and 2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (EMHF) were produced at concentrations up to 4 mg l^–1 and 20 mg l^–1 respectively by yeast fermentation of a heated mixture of a single amino acid and a single sugar added to a yeast extract/peptone/glucose (YPG) medium. About 1 mg DMHF 1^–1 was also produced from precursors in the autoclaved YPG medium but EMHF formation depended entirely on the presence of a heated ribose/amino acid mixture. Glutamate was the best precursor amino acid for both furanones. Formation of EMHF showed a positive, non-linear response to ribose/glutamate concentration from 20 to 200 mM in the heated mixture.     

cfm is a novel gene uniquely expressed in developing forebrain and midbrain, but its null mutant exhibits no obvious phenotype

A novel gene, cfm, that is expressed uniquely during early forebrain and midbrain development was isolated, and its null mutant was generated. cfm does not have any known functional domains, but is conserved in human, chick, Xenopus and zebrafish; a site of phosphorylation by MAP kinase exists in one of the domains conserved among them. Its expression was initially found at the 5-somite stage in the future midbrain and caudal forebrain region. The expression in mesencephalon subsequently decreased, was found in a stripe in the mid mesencephalon at E9.0. The expression in diencephalon was restricted to the dorsal thalamic region by E9.5 and to epiphysis at E12.5. In mouse a cognate, cfm2, exists that is expressed uniquely in the somite just formed and the presomite to be segmented, but not in forebrain or midbrain during early development. However, the cfm null mutant was live-born without any apparent defects.     

A comparative study of the effects of phebrol on the respiratory chains of rat liver, Biomphalaria glabrata and Oncomelania nosophora.

1. Phebrol (sodium 2,5-dichloro-4-bromophenol), a synthetic molluscicide against Oncomelania nosophora, showed a dual effect on rat liver submitochondria, acting as an uncoupler at low concentrations (approximately 10 microM) and an inhibitor of succinate-cytochrome c reductase at high concentrations. 2. Phebrol also inhibited the enzymes responsible for succinate-fumarate conversion, i.e. the succinate-cytochrome c reductase, fumarate reductase and NADH-cytochrome c reductase of the mitochondrial fraction from Biomphalaria glabrata. 3. Kinetic inhibition studies showed succinate-cytochrome c reductase of B. glabrata and O. nosophora to be more sensitive than that of rat liver toward phebrol. 4. Phebrol accumulated in whole tissues of B. glabrata and O. nosophora and had significant effects on the production of succinate, fumarate and malate by these snails. 5. On the basis of these results, the possible sites of inhibition by phebrol of snail respiratory chains are proposed.     

Light-induced enzyme activity changes associated with the photoisomerization of bound spiropyran

A spiropyran compound which undergoes photoisomerization was covalently attached to several enzymes in order to alter their activity by imposing light on them. The enzyme-bound spiropyran exhibited normal photochromism when the matrix enzyme was hydrophobic in nature, whereas reverse photochromism was evident when the spiropyran was attached to hydrophilic enzymes. The activity of the modified enzymes was found to change because of photoisomerization of the bound spiropyran. A fluorescent probe for hydrophobic sites revealed that the modified enzymes remained hydrophobic when the bound spiropyran closed and turned hydrophilic when the spiropyran opened. A mechanism for the photoinduced activity changes is postulated in relation to the enzyme-substrate affinity in the open- and closed-ring states of spiropyran.