Abnormal blood vessel development in mice lacking presenilin-1

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimer's disease. To explore the potential roles of PS1 in vascular development, we examined the vascular system of mouse embryos lacking PS1. PS1-deficient embryos exhibited cerebral hemorrhages and subcutaneous edema by mid gestation. Immunohistochemical analysis revealed vascular remodeling failure in the stomach and trunk dorsal median region of the skin and insufficient formation of the perineural plexus around the spinal cord of the PS1 mutant embryos. The number of capillary sprouting sites reduced and the capillary diameter increased in the mutant brains, especially at the amygdaloid and striatal regions. Endothelial cells in the sprouting capillaries of the mutant mice showed abnormal morphologies such as multiplication, apoptotic and necrotic images, in contrast to pericytes showing a normal appearance. An in vitro assay using para-aortic splanchnopleural mesoderm (P-Sp) revealed aberrant angiogenesis in the explant culture from the mutant. These findings suggest the essential roles of PS1 in angiogenesis.     

Characterization of two cDNA clones encoding isozymes of the F1F0-ATPase inhibitor protein of rice mitochondria

Two cDNA clones encoding F₁F₀-ATPase inhibitor proteins, which are loosely associated with the F₁ part of the mitochondrial F₁F₀-ATPase, were characterized from rice (Oryza sativa L. cv. Nipponbare). A Northern hybridization showed that the two genes (designated as IF ₁ -1 and IF ₁ -2) are transcribed in all the organs examined. However, the steady-state mRNA levels varied among organs. A comparison of the deduced amino acid sequences of the two IF ₁ genes and the amino acid sequence of the mature IF₁ protein from potato revealed that IF₁-1 and IF₁-2 have N-terminal extensions with features that are characteristic of a mitochondrial targeting signal. To determine the subcellular localization of the gene products, the IF₁-1 or IF₁-2 proteins were fused in frame to the green fluorescent protein (GFP) or the fused GFP-β-glucuronidase, and expressed transiently in onion or dayflower epidermal cells. Localized fluorescence was detected in mitochondria, confirming that the two IF₁ proteins are targeted to mitochondria. Received: 9 July 1999 / Accepted: 17 August 1999     

Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion

UDP-L-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that the RHM2/MUM4 gene is involved in conversion of UDP-D-glucose to UDP-L-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-D-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1-370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371-667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-L-rhamnose via an UDP-4-keto-6-deoxy-D-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His(6)-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-D-glucose than for dTDP-D-glucose, the preferred substrate for dTDP-D-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-L-rhamnose, UDP-D-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-D-glucose and UDP-L-rhamnose via feedback inhibition by UDP-L-rhamnose and UDP-D-xylose.     

Liana abundance and relationships to sapling and tree hosts in an East African primary forest

Lianas are an important structural component of tropical rain forests. Recent concern regarding a putative global rise in liana abundance, and its implications for forest conservation, calls for data collection across biomes. We here provide a first assessment and baseline data for a geographical gap in liana surveys to date. We surveyed liana (diameter at breast height [DBH] > 1 cm), tree (DBH > 10 cm) and sapling (DBH ≤ 10 cm) abundance and basal area, as well as liana–host relationships, in a tropical East African primary forest. We recorded a total of 347 liana stems (DBH > 1 cm) in 0.31 ha, with an average basal area of 1.21 m²/ha. Lianas were found to be widespread, with 24% of saplings and 57% of trees colonised by at least one liana, independently of bark texture or host diameter. The dominant liana colonisation strategy was to associate with a single host, through stem twining. We found no evidence of liana density being influenced by host density. We synthesised published liana density data across continents and report that our estimate of liana density for Kibale's primary forest fits within the expected range of liana densities for primary tropical forests. This synthesis further highlights a neotropical sampling bias, which our findings make a step towards addressing.     

Busulfan-induced central polydactyly, syndactyly and cleft hand or foot: a common mechanism of disruption leads to divergent phenotypes

The prevalence of clinical phenotypes that exhibit combinations of central polydactyly, syndactyly, or cleft hand or foot is higher than would be expected for random independent mutations. We have previously demonstrated that maternal ingestion of a chemotherapeutic agent, busulfan, at embryonic day 11 (E11) induces these defects in various combinations in rat embryo limbs. In an effort to determine the mechanism by which busulfan disrupts digital development, we examined cell death by Nile Blue staining and TdT-mediated dUTP nick end labeling (TUNEL) assays; we also carried out whole mount in situ hybridization for fibroblast growth factor-8 (Fgf8), bone morphogenetic protein-4 (Bmp4), and sonic hedgehog (Shh) to examine developmental pathways linked to these defects. In busulfan-treated embryos, diffuse cell death was evident in both ectoderm and mesoderm, peaking at E13. The increased cell death leads to regression of Fgf8 in the apical ectodermal ridge (AER) and Bmp4 and Shh in the underlying mesoderm. The subsequent pattern of interdigital apoptosis and cartilage condensation was variably disrupted. These results suggest that busulfan manifests its teratogenic effects by inducing cell death of both ectoderm and mesoderm, with an associated reduction in tissue and a disruption in the generation of patterning molecules during critical periods of digit specification.     

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5α, a strain that carries EPA biosynthesis genes (pEPAΔ1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.     

Alteration of substrate specificity of fructosyl-amino acid oxidase from <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as -fructosyl -benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.