pH-dependent aggregate forms and conformation of Alzheimer amyloid beta-peptide (12-24)

The conformational transition to a beta-structure and the aggregation process of Alzheimer amyloid beta-peptide (12-24) [abbreviated as A beta(12-24)] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of A beta(12-24) depended on the pH immediately after sample dissolution. Two types of sample dissolution methods, F and R, were employed. For dissolution method F, the peptide sample was immediately dissolved in water and then adjusted to pH 2.2 by adding buffer, while for dissolution method R, the peptide was directly dissolved in the buffer solution. In the latter case, the starting pH was 3.0. Slight fibrils (10-12 nm in diameter) were observed with method F, and wider ribbon-like aggregates (17-20 nm in diameter) with method R, despite the same pH range. A difference between methods F and R was also detected in the CD spectra, especially at pHs near 5.0. The CD intensity of the 214 nm band with method R changed with pH, with the highest value at pH 3.7, whereas that with method F was unchanged at pHs below 5.0. The temperature-dependent CD results showed that a thermostable aggregate of A beta(12-24) occurs at higher pHs than 3.0. NMR analysis showed that deprotonation of the C-terminal carboxylate group in A beta(12-24) triggered the aggregate formation, and the transition from a random coil to a beta-conformation in the C-terminal region of V18-V24 was detected on analysis of the (3)Ja(N) coupling constant in the pH range of 2.2 to 3.0.     

Expression of a soybean nodule‐enhanced phosphoenolpyruvate carboxylase gene that shows striking similarity to another gene for a house‐keeping isoform

Three different cDNAs for phosphoenolpyruvate carboxylase (PEPC) were isolated from soybean root nodules. The full-length cDNA of the most abundant isoform (GmPEPC7) was very similar to another one (GmPEPC15), the nucleotide sequence of which is identical to that of a reported clone (gmppc1) (Vazquez-Tello, A., Whittier, R.F., Kawasaki, T., Sugimoto, T., Kawamura, Y., Shibata, D. (1993) Plant Physiol. 103, 1025–1026). In the coding region, the newly isolated GmPEPC7 and the previously reported were gmppc1 99% and 98% identical at the amino acid and nucleotide levels, respectively. In contrast, they exhibited only 39% identity in the 3′ non-coding region, indicating that they are encoded by distinct genes. Northern blot analysis with 3′ non-coding regions as isoform-specific probes showed that GmPEPC7 is nodule-enhanced whereas GmPEPC15 (gmppc1) is expressed in most soybean tissues. The third clone (GmPEPC4) was much less homologous to the above two clones and thus was not further characterized. It was also shown by in situ hybridization that the nodule-enhanced isoform is expressed in all cell types in nodules, including in Bradyrhizobium-infected and uninfected cells and cortical cells. A relatively strong hybridization signal was detected in the vascular bundle pericycle. Southern blot analysis indicated that there are only two PEPC genes exhibiting a high degree of similarity in the soybean genome, one for the nodule-enhanced GmPEPC7 and the other for the constitutively expressed gmppc1. A phylogenetic tree based on the amino acid sequences of soybean PEPCs and nodule-enhanced PEPCs of alfalfa and pea suggested that the soybean nodule-enhanced isoform evolved from the housekeeping PEPC gene after the ureid-translocating and amide-translocating legumes diverged from each other.     

Photosynthesis–nitrogen relationships in species at different altitudes on Mount Kinabalu, Malaysia

In situ photosynthetic nitrogen-use efficiency (PNUE, photosynthetic capacity per unit leaf nitrogen) was investigated in species that commonly distributed at different altitudes (600–3700m above sea level) on Mount Kinabalu. Photosynthetic nitrogen-use efficiency was lower in species at higher altitudes, with a mean PNUE at 3700m being one-third as large as that at 600m. This difference in PNUE was larger than that explained by the biochemical response to lower air pressures only. Across altitudes a negative correlation between ¹³C abundance (¹³C) and PNUE was found. Species at higher altitudes tended to have higher ¹³C, suggesting that they had a lower conductance for CO₂ diffusion from the air to chloroplasts. The lower conductance might be responsible for the lower PNUE in species at higher altitudes. Although leaf nitrogen content per unit area tended to be higher at higher altitudes, it did not seem to contribute to increasing photosynthetic rates. Thus, the idea that a higher nitrogen content at higher altitudes is a compensation for a lower PNUE was not supported. In contrast to the large difference in PNUE among altitudes, PNUE tended to converge within a narrow range among species growing at the same altitude.     

Novel preparation method for surfactant-coated enzymes using W/O emulsion

Summary A novel preparation method for surfactant-coated enzymes has been developed using a W/O emulsion. The enzymatic activity of chymotrypsin in isooctane significantly increased with the coating of surfactants. The surfactant-coated chymotrypsin showed a high enzymatic activity for amidation, although powdered chymotrypsin did not show the activity. Further, the coated enzyme showed a remarkably high storage stability.     

Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H

To construct a DNA-linked RNase H, which cleaves RNA site-specifically at high temperatures, the 15-mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA (PPT-RNA), was cross-linked to the unique thiol group of Cys135 in the Thermus thermophilus RNase HI variant. The resultant DNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH, in which the RNase H portion of the d15-C135/TRNH is replaced by the Escherichia coli RNase HI variant, cleaved the 15-mer PPT-RNA site-specifically. The mixture of the unmodified enzyme and the unlinked 15-mer DNA also cleaved the PPT-RNA but in a less strict manner. In addition, this mixture cleaved the PPT-RNA much less effectively than the DNA-linked enzyme. These results indicate that the cross-linking limits but accelerates the interaction between the enzyme and the DNA/RNA substrate. The d15-C135/TRNH cleaved the PPT-RNA more effectively than the d15-C135/ERNH at temperatures higher than 50 degrees C. The d15-C135/TRNH showed the highest activity at 65 degrees C, at which the d15-C135/ERNH showed little activity. Such a thermostable DNA-linked RNase H may be useful to cleave RNA molecules with highly ordered structures in a sequence-specific manner.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

How social relationships influence a monkey's choice of feeding sites in the troop of Japanese macaques (Macaca fuscata fuscata) on Koshima islet

When the individual Japanese macaques of the Koshima troop feed on natural food, they usually feed alone. In situations where animals usually feed without other animals, there is a possibility that subordinate animals may avoid feeding sites at which dominant animals are feeding. This paper examines whether social relationships such as kinship or dominance exert any influence on an animal's choice of feeding sites, by analyzing episodes in which an animal approached and climbed into a tree where other animals were. As a result, it was found that social relationships did not influence whether an animal climbed into a tree where other animals were feeding, and that no particular age-sex pair co-fed. Agonistic interactions frequently occurred when the inter-individual distance was less than 1 m. From these findings, the feeding sites were divided into two spaces: (1) a tolerance feeding space, and (2) an intolerance feeding space. It is presumed that animals can feed without entering others' intolerance feeding spaces when food is abundant, as it was in the present study period. Thus social relationships do not influence an animal's choice of feeding sites in such a situation.     

Production of mannosylerythritol lipids as biosurfactants by resting cells ofCandida antarctica

Summary The resting cells ofCandida antarctica strain T-34 was found to produce a large amount of mannosylerythritol lipids as biosurfactants when incubated in the medium containing only the carbon source. The resting cells prepared from different water-soluble carbon sources were able to produce the lipids abundantly from water-insoluble carbon sources. Under the optimal conditions in a shake culture, the concentration of the total lipids amounted to about 47 g/l after 6 days, and the yield of the lipids became higher than that obtained by using the growing cells of the strain.