d-Ser-containing humanin shows promotion of fibril formation

Humanin (HN), a peptide of 24 amino acid residues, suppresses the neuronal cell death that is induced by the gene products of Alzheimer’s disease. HN contains two Ser residues at positions 7 and 14. Because the proportion of d-Ser isomerized from l-Ser in proteins appears to increase as cellular organs age, we explored the structural effects of the isomerization of each Ser residue in HN. By using a thioflavin-T assay to detect fibril formation, we found that an HN derivative that contained two isomerized d-Ser residues had a greater tendency to form fibrils than did wild-type HN or HNs containing single d-Ser residues. A previous report showed that HN containing two d-Ser residues exerts neuroprotective activity. Our data, therefore, suggest that the fibril formation by HN that contains two d-Ser residues may promote HN neuroprotective activity.     

Habitat associations with topography and canopy structure of tree species in a tropical montane forest on Mount Kinabalu,Borneo

Habitat associations with topography and canopy structure of 42 abundant tree species were studied in a 2.74-ha plot of tropical montane forest on Mount Kinabalu, Borneo. Many of these species belong to the same higher taxa including eight families and four genera. Analysis of intraspecific spatial distributions for stems ≥ 10 cm diameter revealed that 28 species (including all six species of Fagaceae) showed aggregated distributions at the 100-m² and/or 400-m² scales, and that 20 species showed habitat associations with topography by torus-translation tests; 17 species showed both characteristics. Species' associations with the local canopy structure were characterized by crown position index (CPI), which was defined relative to neighbour trees. The CPI differed greatly among individual stems at 10–40 cm diameter, and 19 species showed significantly different frequencies of crowns exposed vertically versus those shaded beneath the canopy. Mean growth rates at 10–40 cm diameter and size distributions of species were not related to topographic associations, but were explained by the associations with canopy structure; species with more exposed crowns grew faster and had less positively skewed distributions. Diversity in habitat associations was manifest between two genera (Syzygium and Tristaniopsis) in the family Myrtaceae and among species in these genera, but was less evident in other families and two genera (Garcinia and Lithocarpus). This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphorus and nitrogen resorption from different chemical fractions in senescing leaves of tropical tree species on Mount Kinabalu,Borneo

Nutrient resorption, a process by which plants degrade organic compounds and resorb their nutrients from senescing tissues, is a crucial plant function to increase growth and fitness in nutrient-poor environments. Tropical trees on phosphorus (P)-poor soils are particularly known to have high P-resorption efficiency (PRE, the percentage of P resorbed from senescing leaves before abscission per total P in green leaves). However, the biochemical mechanisms underlying this greater PRE remain unclear. In this study, we determined the P concentration in easily soluble, nucleic acid, lipid and residual fractions for green and senescent leaves of 22 tree species from three sites, which differed in P availability, on the lower flanks of Mt. Kinabalu, Borneo. PRE varied from 24 to 93% and was higher in species from the P-poor site. P-resorption rate was greatest from the lipid fraction, the nucleic acid fraction, and lowest in the easily soluble fraction and the residual fraction when all the species were pooled. For species with higher PRE, P-resorption rate of the residual fraction was relatively high and was comparable in magnitude to that of the other labile fractions. This suggests that tree species inhabiting P-poor environments increased PRE by improving the degradation of recalcitrant compounds. This study suggests that plants selectively degrade organic compounds depending on environmental conditions, which is a key mechanism underlying the variation of PRE.     

Isolation of a DNA-binding protein from Deinococcus radiodurans having an affinity for a Z-form polynucleotide

A protein which preferentially binds Z-form duplex DNA has been purified from the cells of Deinococcus radiodurans. The molecular weight of the protein was estimated to be approximately 68,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. Amino acid analysis of the protein indicates that it is not so basic since it contains a lower mole percent of lysine and higher mole percent of aspartic acid than those in histone-like DNA binding protein II (HU) of Escherichia coli. The first fifteen amino acid residues from the N-terminus have been also determined.     

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.     

Adaptive repair of cross-links in DNA of Micrococcus radiodurans

Cross-links in the DNA of Micrococcus radiodurans induced by mitomycin C were repaired during post-incubation. This repair process was inhibited in cells post-incubated in the presence of chloramphenicol. However, the removal of cross-links in DNA was almost normal, even in the presence of chloramphenicol, if the cells were pretreated with lower concentrations of mitomycin C.     

The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Δ yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (γ-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced γ-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.     

Embryonic stem cell differentiation system for evaluating gene functions involved in physiological megakaryocytic differentiation

Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP(+) cells among CD41(+) cells). Insertion of full-length or small core β-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (～60% GFP(+) cells among CD41(+) cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.