Isolation and characterization of 20 microsatellite loci for an important tropical tree Shorea leprosula (Dipterocarpaceae) and their applicability to S. parvifolia

Shorea leprosula is an important timber tree in Southeast Asia. From a genomic library enriched for dinucleotide (CT) repeats, 20 polymorphic microsatellite markers were developed for this species. Polymorphism of these loci was evaluated in a sample of 24 adult individuals from a natural population. The number of alleles ranged from five to 15, and the observed heterozygosity ranged from 0.333 to 0.875. Probability of paternity exclusion, in the case where the mother is known, ranged from 0.285 to 0.784. These markers were also characterized for applicability in S. parvifolia; 16 loci were successfully cross‐amplified and showed high levels of polymorphism.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

PDZ interaction sites in integrin alpha subunits. T14853, TIP/GIPC binds to a type I recognition sequence in alpha 6A/alpha 5 and a novel sequence in alpha 6B

We used published peptide library data to identify PDZ recognition sequences in integrin alpha subunit cytoplasmic domains and found that the alpha(6)A and alpha(5) subunits contain a type I PDZ binding site (TSDA*) (asterisk indicates the stop codon). The alpha(6)A cytoplasmic domain was used for screening a two-hybrid library to find interacting proteins. The bulk of the captured cDNAs (60%) coded for TIP-2/GIPC, a cytoplasmic protein with one PDZ domain. The interaction of TIP-2/GIPC with different integrin subunits was tested in two-hybrid and in vitro binding assays. Surprisingly, TIP-2/GIPC bound strongly to the C terminus of both alpha(6)A and alpha(6)B, although the alpha(6)B sequence (ESYS*) is not suggestive of a PDZ binding site because of its polar C-terminal residue. For high affinity interaction with TIP-2/GIPC, at least one of the residues at positions -1 and -3 must be negatively charged. An aliphatic residue at position 0 increases the affinity of but is not required for this interaction. The alpha(5) integrin subunit also bound to TIP-2/GIPC. The alpha(6) integrin and TIP-2/GIPC co-localize in retraction fibers in carcinoma cells plated on laminin, a finding suggesting a functional interaction in vivo. Our results demonstrate that both splice variants of alpha(6) integrin contain a conserved PDZ binding site that enables interaction with TIP-2/GIPC. The binding site in alpha(6)B defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.     

Chlamydocin analogues from the soil fungus Peniophora sp.: structures and plant growth-retardant activity

Three chlamydocin analogues that show activity for reducing the height of rice seedlings without blotch and wilting were isolated from the culture filtrate of the soil fungus Peniophora sp. and were characterized by spectral data and chemical conversion. For two of these, it is their first isolation as natural products. The proposed role of the functional groups of the 2-amino-8-oxodecanoic acid moiety of these analogues is discussed in terms of growth retardant activity.     

Characterization of an outer membrane protein gene, pgmA, and its gene product from Porphyromonas gingivalis

A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses. Therefore, the gene (formerly ORF5) was designated pgmA, the P. gingivalis outer membrane protein A gene. The gene product, PgmA, was sensitive to protease, and was detected as a 60-kDa protein from wild-type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease-deficient mutants without this treatment prior to electrophoresis. PgmA was indeed present in the membrane fraction. Its nature was determined to be that of outer membrane proteins in gram-negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents. No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.     

Integrin alpha4beta1-dependent adhesion to ADAM 28 (MDC-L) requires an extended surface of the disintegrin domain

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.     

Heme-Cu complexes as oxygen-activating functional models for the active site of cytochrome c oxidase

Tri(2-pyridylmethyl)amineCu complex-linked iron meso-tetraphenylporphyine derivatives were prepared to model the active site of cytochrome c oxidase. Exposure to oxygen converted the reduced forms of the complexes to the corresponding stable mu-peroxo species in spite of the presence of three coordination sites, two on the heme and one on the Cu. The oxy forms were characterized spectroscopically. Kinetic analyses of the oxygenation reactions of the reduced forms suggests that preferential O2 binding occurs at the Cu site over the heme. This mechanism is also supported by examination of the redox potentials of the two metal ions. Since the peroxy complexes of the models exhibit a structure similar to that of the previously reported fully-oxidized form, the relevance of the model chemistry to the enzyme reaction is discussed.     

Wax ester production from n-alkanes by Acinetobacter sp. strain M-1: ultrastructure of cellular inclusions and role of acyl coenzyme A reductase

Acinetobacter sp. strain M-1 accumulated a large amount of wax esters from an n-alkane under nitrogen-limiting conditions. Under the optimized conditions with n-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis.     

Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy

We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equipping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level.