BMP and Hh signaling affects primordial germ cell division in Drosophila

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up- and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.     

A simple purification method for the preparation of solubilized chlorophyllase from Chlorella protothecoides

A simple and rapid purification procedure is described for the routine preparation of large quantities of purified chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from Chlorella protothecoides. The enzyme with specific activity of 960 nmol chlorophyll a hydrolyzed (mg protein)^?1 min^?1 was prepared by treating the homogenate with n-butanol, ammonium sulfate fractionations and gel filtration through Sephadex G-200 and Sepharose CL-6B, with a yield of 53% of activity based on the butanol extract. The enzyme preparation showed apparent homogeneity as judged by polyacrylamide gel electrophoresis. The procedures take only 4 days and can be operated routinely without column repacking.     

An inter-subunit disulfide bond affects affinity of human lung extracellular superoxide dismutase to heparin

Human extracellular superoxide dismutase (EC-SOD) was purified to homogeneity from lung tissue and the nature of the binding of heparin to EC-SOD was investigated. The enzyme was purified using three column chromatographic steps, and 127 μg of purified EC-SOD was obtained. A specific anti-human EC-SOD antibody was obtained by immunization with the purified enzyme. Western blot analysis of the heparin affinity chromatography product indicated that the presence of the inter-subunit disulfide bond affects the affinity of EC-SOD for heparin. The affinity of EC-SOD for heparin is a very important feature of the enzyme because it controls the distribution of the enzyme in tissues. The present study suggests that, not only the processing of the C-terminal region but inter-subunit disulfide bonds also play a role in determining the tissue distribution of EC-SOD. Moreover, the results obtained here also suggest that the redox state of the tissues might regulate the function of the EC-SOD.     

Mitochondrial ATPase subunit 6 and cytochrome B gene polymorphisms in young obese adults.

We hypothesized that the mutational strand asymmetry is more strongly exerted upon the mitochondrial cytochrome b (Cytb) gene, which is distant from the origin of the light-strand replication (Ori(L)), than upon the ATPase subunit 6 (ATP6) gene, which is close to the Ori(L). To test this hypothesis, we determined the sequences of these two genes in 96 Japanese young obese adults. The frequency of G-->A transitions was significantly higher than that of C-->T transitions in the Cytb gene, whereas the frequencies of G-->A and C-->T transitions were not significantly different in the ATP6 gene. The marked mutational strand asymmetry in the Cytb gene can be explained by the deamination of C to uracil in the long single-stranded state of the heavy strand during replication. The ratio of the nonsynonymous substitutions at the second codon positions to those at the first codon positions was significantly lower in the Cytb gene than in the ATP6 gene. The physicochemical differences between the standard and the replaced amino acid residues were significantly smaller in the Cytb gene than in ATP6 one. The present study indicates that amino acid sequences are less variable for Cytb than for ATP6 in spite of the strong mutational strand asymmetry for the Cytb gene.     

Novel Subtype of Peroxisomal Acyl-CoA Oxidase Deficiency and Bifunctional Enzyme Deficiency with Detectable Enzyme Protein: Identification by Means of Complementation Analysis

We describe four infants with a novel subtype of an isolated deficiency of one of the peroxisomal β-oxidation enzymes with detectable enzyme protein. The patients showed characteristic clinical and biochemical abnormalities, including hypotonia, psychomotor retardation, hepatomegaly, typical facial appearance, accumulation of very-long-chain fatty acids, and decreased lignoceric acid oxidation. However, β-oxidation enzyme proteins were detected by immunoblot analyses, and large peroxisomes were identified by immunofluorescence staining. In order to identify the underlying defect in these patients, complementation analysis was introduced using fibroblasts from these patients and patients with an established deficiency of either acyl-CoA oxidase or bifunctional enzyme, as identified by immunoblotting. In the complementing combinations, fused cells showed increased lignoceric acid oxidation, resistance against 1-pyrene dodecanoic acid/UV selection, and normalization of the size and the distribution of peroxisomes. The results indicate that two patients with a more severe clinical course were suffering from bifunctional enzyme deficiency and that the other two infants, who were siblings and had a less severe clinical presentation, were the first patients with acyl-CoA oxidase deficiency with detectable enzyme protein.     

Quasispecies of TT Virus (TTV) with Sequence Divergence in Hypervariable Regions of the Capsid Protein in Chronic TTV Infection

Three hypervariable regions were identified in a central portion of open reading frame 1 of TT virus DNA, which codes for a putative capsid protein of 770 amino acids. TT virus circulates as quasispecies, with many amino acid substitutions in hypervariable regions, to evade immune surveillance of the hosts and to establish a persistent infection.     

Gurmarin suppression of licking responses to sweetener-quinine mixtures in C57BL mice

Gurmarin (Gur) is a peptide that selectively suppresses responses of the chorda tympani nerve to sweet substances in rats and mice. In the present study, we examined the effect of Gur on behavioral responses to sweet substances in C57BL mice. To accomplish this, we developed a new short-term lick test and measured numbers of licks for 10 s for sweet substances mixed with quinine hydrochloride (QHCl) in water-deprived mice. Numbers of licks for sucrose mixed with 1 or 3 mM QHCl increased with increasing concentration of sucrose from 0.01 to 1.0 M. Oral infusion with 30 micro g/ml Gur produced significant decreases in responses to concentration series for sucrose mixed with 3 mM QHCl, whereas no such effect by Gur was observed in responses to QHCl alone or QHCl-mixed HCl, NaCl or monosodium glutamate. The Gur suppression of QHCl-mixed sucrose responses, which otherwise lasted for 2-3 h, rapidly returned to approximately 80% of control levels after oral infusion with beta-cyclodextrin. These results are comparable to neural data previously found in chorda tympani responses, and thereby provide further evidence for Gur as a sweet response inhibitor in C57BL mice. In the other aspect, our newly developed short-term test can also provide a tool for measurements of taste-guided behavioral responses to sweeteners.     

Formation and Degradation of Protochlorophylls in Etiolated and Greening Cotyledons of Cucumber

The formation, degradation and phototransformation of protochlorophylls(Pchls) in the etiolated and greening cotyledons of cucumber(Cucumis sativus L.) werestudied using high-performance liquidchromatography. The pigment analysis of etiolated cotyledonsshowed the presence of four Pchls esterified with phytol, tetrahydrogeranylgeraniol(THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG).The content of Pchl THGG rapidly increased during dark developmentof seedlings and reached a maximal level at 4th day, then decreasedgradually. Unlike Pchl THGG, Pchl DHGG and Pchl GG showed asmall peak at 3rd day followed by a one-day lag, then accumulationbegan. The content of Pchl DHGG reached a maximal level at 12thday, then decreased rapidly, while Pchl GG continued to increaseand its maximal stage was not attained at 15th day. The contentof Pchl phytol remained very low during dark growth. These resultsmay indicate that with increasing age, the inactivation of hydrogenationof the alcohol moiety of Pchl proceeds stepwise at the sitesof Pchl THGG, Pchl DHGG and Pchl GG, in that order, withoutaffecting the esterification of Pchlide. The content of four Pchls remained unchanged before and after30-s illumination, indicating that none of the four Pchls istransformed to chlorophyll by light. Under continuous illumination,Pchls decreased exponentially or linearly at a rather slow rate.Thus, the four Pchls are not direct precursors for chlorophylland are metabolized slowly under greening. (Received December 6, 1982; Accepted April 13, 1983)     

Lipid-binding form is a key conformation to induce a programmed cell death initiated in tobacco BY-2 cells by a proteinaceous elicitor of cryptogein

Cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea , induces a remarkable hypersensitive cell death in tobacco cells. Two cryptogein mutants were analysed to characterize the induction mechanism of cell death; one was a newly synthesized mutant N93A whose 93rd Asn residue was changed to Ala, the other was K13V whose Lys at position 13 was replaced with Val. The effect of these mutations was evaluated in terms of extracellular alkalization, production of active oxygen species (AOS) and progression to death. The mutation N93A resulted in a reduction in activity to 71.0, 74.6 and 24.5% for original rates of extracellular alkalization, AOS production and cell death progression, respectively. In the case of the K13V mutation, these rates changed to 114, 3.38 and 7.40%, respectively. The lipid-binding activities of the mutants were analysed using fluorogenic lipid of dehydroergosterol. The results for N93A and K13V were 38.3 and 3.40% compared with the wild type, respectively. These findings indicate that the lipid-binding form was the only conformation to induce the production of AOS and programmed cell death in plants.     

Radial Columnar Patches in the Chimeric Cerebral Cortex Visualized by Use of Mouse Embryonic Stem Cells Expressing β-Galactosidase

Presumed radial migration of neuroblasts from the ventricular to pial surface during formation of the cerebral cortex predicts radial columnar patches in chimeric brains. Lack of adequate cell marker for neurons, however, has hindered such chimera analysis. We used a mouse embryonic stem cell line expressing β-galactosidase gene to produce chimeric brains. Patches of the labeled cells were examined by whole mount staining and also by computer-assisted three-dimensional reconstruction from serial paraffin sections. Our study revealed presence of coherent radial columnar patches in the prenatal cerebral cortex, thus giving a direct evidence for the radial migration of neurons. These columnar patches were less clear in adult brains, suggesting cell mixing during later development and maturation.