The iron content of iron superoxide dismutase: determination by anomalous scattering

The number of iron atoms in the dimeric iron-containing superoxide dismutase from Pseudomonas ovalis and their atomic positions have been determined directly from anomalous scattering measurements on crystals of the native enzyme. To resolve the long-standing question of the total amount of iron per molecule for this class of dismutase, the occupancy of each site was refined against the measured Bijvoet differences. The enzyme is a symmetrical dimer with one iron site in each subunit. The iron position is 9 A from the intersubunit interface. The total iron content of the dimer is 1.2 +/- 0.2 moles per mole of protein. This is divided between the subunits in the ratio 0.65:0.55; the difference between them is probably not significant. Since each subunit contains, on average, slightly more than half an iron atom we conclude that the normal state of this enzyme is two iron atoms per dimer but that some of the metal is lost during purification of the protein. Although the crystals are obviously a mixture of holo- and apo-enzymes, the 2.9 A electron density map is uniformly clean, even at the iron site. We conclude that the three-dimensional structures of the iron-bound enzyme and the apo-enzyme are identical.     

Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds

Thirty-five derivatives of cinnamic acid and related compounds were tested for inhibition against phenylalanine ammonia-lyase (PAL) derived from sweet potato, pea and yeast. Caffeic and gallic acids showed inhibition against PAL originating from higher plants, but not against yeast PAL. In contrast, yeast PAL was specifically inhibited by p-hydroxycinnamic and p-hydroxybenzoic acids. The results suggest that caffeic and gallic acids may act as regulatory substances in phenylpropanoid metabolism in higher plants. Inhibition experiments with synthetic cinnamic acid derivatives have revealed that the presence of a hydrophobic aromatic ring, α,β-double bond and carboxyl group is essential for inhibitory activity. 2-Naphthoic acid which fulfills these structural requirements showed a strong inhibition. The size and shape of the active site is discussed from structure-activity relationships of cinnamic acid derivatives. o-Chlorocinnamic acid, one of the strongest inhibitors found in this study showed an inhibitory effect on the growth of the roots of rice seedlings.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.     

Scaling of Petiole Dimensions with Respect to Leaf Size for a Tropical Tree, Scaphium macropodum (Sterculiaceae), in Borneo

Scaphium macropodum (Miq.) Beumee ex Heyne (Sterculiaceae) in a tropical rain forest in West Kalimantan (Indonesia) was analyzed from the viewpoint of statics. The petiole diameter must increase with increasing leaf size to retain enough mechanical stability and a sufficient amount of conductive vessels. The petiole's cross-sectional area at its base was found to be proportional to the leaf blade's dry mass, which indicates that Shinozaki's pipe model is applicable to leaves with different sizes. Although larger leaves produce greater bending moments on the petiole's cross-section as a result of their greater weights, the bending stresses at the petiole's base caused by the leaf's weight were constant at ca. 76,900 g cm⁻² regardless of leaf size. Thicker petioles increase the leaf's mechanical stability, but require sizable energy investments for their construction. It is hypothesized that the constant value for petiolar stress indicates an optimal balance between energy economy and the mechanical stability of S. macropodum leaves. To keep bending stress constant, the leaf blade's center of gravity shifts to a more proximal position and the cross-sectional area of the petiole increases. Received 8 December 1997/ Accepted in revised form 1 December 1998     

Sex ratio and gender-dependent neighboring effects in <Emphasis Type="Italic">Podocarpus nagi</Emphasis>, a dioecious tree

We analyzed sex ratio, growth rates, and spacing among individuals of Podocarpus nagi, a dioecious tree, on Mt. Mikasa, Nara City, Japan. The sex ratio of reproductive trees ≥ 5 cm in stem diameter at breast height (dbh, 130 cm above ground level) was significantly male-biased. The sex ratio was male-biased in the < 20 cm and ≥ 50 cm size classes, while it did not depart from 1:1 in the 20 ≤ dbh < 50 cm class. Growth rate varied with tree size in males but not in females. The precocity and vigor of males suggests that differences in reproductive costs between sexes induce the biased sex ratio. Random labeling tests on the positions of reproductive trees showed that in the < 30 cm class, males and females were distributed randomly and independently from each other. In the ≥ 30 cm class, males were significantly clumped, whereas females were randomly distributed. Males and females showed significant repulsion, i.e., a spatial segregation of sexes. Both intra- and intersexual effects on the growth rate of crowding by neighbors were significant for females, but not for males. Maximum competitive interference was observed at a distance of 5 m, which corresponded approximately to the radius of clumps of large males and to the significant repulsive distance between large males and females. These results suggest that sexual differences in sensitivity to local crowding are related to the formation of gender-dependent spatial patterns. Formation of female-repulsive male clumps and a male-biased sex ratio may intensify the decreased probability of regeneration near males, as suggested by the limited seed-dispersal range of this species, thereby promoting coexistence with other species.     

Tree allometry and improved estimation of carbon stocks and balance in tropical forests

Tropical forests hold large stores of carbon, yet uncertainty remains regarding their quantitative contribution to the global carbon cycle. One approach to quantifying carbon biomass stores consists in inferring changes from long-term forest inventory plots. Regression models are used to convert inventory data into an estimate of aboveground biomass (AGB). We provide a critical reassessment of the quality and the robustness of these models across tropical forest types, using a large dataset of 2,410 trees ≥ 5 cm diameter, directly harvested in 27 study sites across the tropics. Proportional relationships between aboveground biomass and the product of wood density, trunk cross-sectional area, and total height are constructed. We also develop a regression model involving wood density and stem diameter only. Our models were tested for secondary and old-growth forests, for dry, moist and wet forests, for lowland and montane forests, and for mangrove forests. The most important predictors of AGB of a tree were, in decreasing order of importance, its trunk diameter, wood specific gravity, total height, and forest type (dry, moist, or wet). Overestimates prevailed, giving a bias of 0.5–6.5% when errors were averaged across all stands. Our regression models can be used reliably to predict aboveground tree biomass across a broad range of tropical forests. Because they are based on an unprecedented dataset, these models should improve the quality of tropical biomass estimates, and bring consensus about the contribution of the tropical forest biome and tropical deforestation to the global carbon cycle. Electronic Supplementary Material Supplementary material is available for this article at     

Mitochondrial peroxiredoxin-3 protects hippocampal neurons from excitotoxic injury in vivo

Mitochondria are involved in excitotoxic damage of nerve cells. Following the breakdown of the calcium-buffering ability of mitochondria, mitochondrial calcium overload induces reactive oxygen species (ROS) bursts that produce free radicals and open permeability transition pores, ultimately leading to neuronal cell death. In the present study, we focused on a mitochondrial antioxidant protein, peroxiredoxin-3 (Prx-3), to investigate the mechanism by which toxic properties of ROS were up-regulated in mitochondria of damaged nerve cells. Immunohistochemical analysis revealed that Prx-3 protein exists in mitochondria of rat hippocampus, whereas we found a significant decrease in Prx-3 mRNA and protein levels associated with an increase in nitrated proteins in the rat hippocampus injured by microinjection of ibotenic acid. Furthermore, in vivo adenoviral gene transfer of Prx-3 completely inhibited protein nitration and markedly reduced gliosis, a post-neuronal cell death event. Since mitochondrial Prx-3 seems to be neuroprotective against oxidative insults, our findings suggest that Prx-3 up-regulation might be a useful novel approach for the management of neurodegenerative diseases.     

Crystallization and preliminary X-ray crystallographic analysis of plant ferritin from Glycine max

The iron storage protein ferritin from soybean (Glycine max) was expressed in E. coli and crystallized using the hanging drop vapor diffusion method with sodium tartrate as the precipitant. The crystals belong to the tetragonal I4(1)22 space group, with unit cell parameters a=b=324.0, c=182.7 A. The diffraction data were collected up to a resolution of 3.0 A with a multi-wire area detector.