Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells

Fucoidan from Cladosiphon okamuranus and its sulfate derivatives were prepared. Sulfate contents of native and oversulfated fucoidan were estimated to be 13.5% and 32.8%, respectively. The results of (1)H NMR suggest that 2,4-di-O-sulfo-, 2-mono-O-sulfo- and 4-mono-O-sulfo-l-fucopyranose were involved in oversulfated fucoidan and 4-mono-O-sulfo-l-fucopyranose was involved in native fucoidan. The oversulfated fucoidan reduced the proliferation of U937 cells in a dose-dependent manner, but the activity of native fucoidan was weak. The sulfate content and substituting position of sulfate group might be important factors of anti-proliferative activity in U937 cells. To examine whether the anti-proliferative activity of oversulfated fucoidan was caused by induction of apoptosis, apoptosis assay, caspase-3 activity assay and Western blotting analysis were performed. These results indicated that the oversulfated fucoidan induced apoptosis via caspase-3 and -7 activation-dependent pathway.     

Gallotannins of the freshwater green alga Spirogyra sp.

The green alga Spirogyra sp. accumulates tetra- through undecagalloylglucosyl gallotannins. The hexa- through undecagalloylglucoses are predominantly based on 1,2,3,4,6-penta-O-galloylglucose, whereas the major pentagalloylglucose is 3-O-digalloyl-1,2,6-tri-O-galloyl-β-D-glucose.     

Structure and antiherpetic activity among the Tannins

In order to investigate the relationship between the antiherpetic activity and the structure of tannins, the activities of 38 such compounds were examined. The results indicate that the activities of hydrolysable tannins were dependent on the number of galloyl or hexahydroxydiphenoyl groups and those of condensed ones on the degree of condensation. On the other hand, the more active tannins were the wore cytotoxic.     

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product.

Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is unique among those of alpha-amylase family enzymes due to a loop (residues 193-218) that is located at the end of the cleft around the nonreducing region and forms a 'dam'-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis.     

Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63

Proteins belonging to the glycoside hydrolase family 63 (GH63) are found in bacteria, archaea, and eukaryotes. Eukaryotic GH63 proteins are processing α-glucosidase I enzymes that hydrolyze an oligosaccharide precursor of eukaryotic N-linked glycoproteins. In contrast, the functions of the bacterial and archaeal GH63 proteins are unclear. Here we determined the crystal structure of a bacterial GH63 enzyme, Escherichia coli K12 YgjK, at 1.78 Å resolution and investigated some properties of the enzyme. YgjK consists of the N-domain and the A-domain, joined by a linker region. The N-domain is composed of 18 antiparallel β-strands and is classified as a super-β-sandwich. The A-domain contains 16 α-helices, 12 of which form an (α/α)₆-barrel; the remaining 4 α-helices are found in an extra structural unit that we designated as the A′-region. YgjK, a member of the glycoside hydrolase clan GH-G, shares structural similarity with glucoamylase (GH15) and chitobiose phosphorylase (GH65), both of which belong to clan GH-L. In crystal structures of YgjK in complex with glucose, mannose, and galactose, all of the glucose, mannose, and galactose units were located in the catalytic cleft. YgjK showed the highest activity for the α-1,3-glucosidic linkage of nigerose, but also hydrolyzed trehalose, kojibiose, and maltooligosaccharides from maltose to maltoheptaose, although the activities were low. These findings suggest that YgjK is a glucosidase with relaxed specificity for sugars.     

Early self-regulatory mechanisms control the magnitude of CD8+ T cell responses against liver stages of murine malaria

Following immunization with Plasmodium yoelii sporozoites, the CD8(+) T cell population specific for the SYVPSAEQI epitope expressed in sporozoite and liver stages of this malaria parasite revealed the existence of a short term Ag presentation process that translated into a single clonal burst. Further expansion of this CD8(+) T cell population in conditions of sustained Ag exposure and additional supply of naive cells was inhibited by regulatory mechanisms that were developed as early as 24-48 h after priming. Studies using mouse models for Plasmodium or influenza virus infections revealed that this mechanism is Ag specific and is mediated by activated CD8(+) T cells that inhibit the priming of naive cells. This interference of the priming of naive cells appeared to result from limited access to Ag-presenting dendritic cells, which become disabled or are eliminated after contact with activated cells. Thus, concomitantly with the development of their effector antimicrobial capacity, CD8(+) T cells also acquire a self-regulatory role that is likely to represent one of the earliest mechanisms induced in the course of an immune response and that limits the magnitude of the early expansion of CD8(+) T lymphocytes reactive to microorganisms.     

Cloning and characterization of the homolog of mammalian lipopolysaccharide-binding protein and bactericidal permeability-increasing protein in rainbow trout Oncorhynchus mykiss

We cloned two cDNAs denoted as RT-LBP/BPI-1 and RT-LBP/BPI-2, respectively, which were derived from the mRNA of head kidney from rainbow trout. They showed structural homology with LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) in mammals. The full-length cDNA of RT-LBP/BPI-1 and RT-LBP/BPI-2 is 1666 and 1741 bp, respectively. Both cDNAs encoded 473 aa residues, including the amino acids conserved in mammalian LBP and BPI proteins that were assumed to be involved in LPS binding. The overall coding sequence of RT-LBP/BPI-1 has 33% amino acid homology to human LBP and 34% to human BPI, and RT-LBP/BPI-2 has 32% amino acid homology to human LBP and 33% to human BPI. Three-dimensional structure analysis by three-dimensional/one-dimensional (3D-1D) methods also demonstrated that RT-LBP/BPI-1 and RT-LBP/BPI-2 proteins showed significant similarity to human BPI, having a boomerang shape with N-terminal and C-terminal barrels. Phylogenetic analysis showed that the LBP and BPI genes seemed to be established after the divergence of mammals from teleosts. These results suggested that RT-LBP/BPI-1 and RT-LBP/BPI-2 may be a putative ortholog for mammalian LBP and/or BPI genes. This is the first study to identify the LBP family genes from nonmammalian vertebrates.     

Confirmation by DNA analysis that Contarinia maculipennis (Diptera: Cecidomyiidae) is a polyphagous pest of orchids and other unrelated cultivated plants

The cytochrome oxidase subunit I gene in mitochondrial DNA of 53 larvae of Contarinia maculipennis Felt from flower buds of various host plants, collected from Hawaii, Japan and Thailand was analysed. Monophyly of the clade including C. maculipennis from Hawaii, Thailand and Japan was supported. There was no sequential variation within the specimens from Hawaii and Japan, which differed from one another by 6 bp (1.37%). Three haplotypes were recognized in specimens from Thailand but differences from Hawaiian and Japanese specimens were small. Overall, there were no differences in the 146 deduced amino acid residues. It is therefore concluded that C. maculipennis is a polyphagous species that can develop on plant hosts representing at least seven botanical families. This pest of Dendrobium flower buds in glasshouses is considered to have entered Hawaii, Florida and Japan from Southeast Asia, and was recently intercepted in the Netherlands. Infestations have established and spread in orchid glasshouses, causing concern about the possibility of more extensive damage to orchids and to crops, such as bitter gourd, grown in close proximity to orchid glasshouses in Japan. The potential usefulness of DNA analysis in determining host plant ranges of morphologically identical cecidomyiid species that are currently identified solely on differences of host plant is emphasized.     

IL-4-secreting CD4+ T cells are crucial to the development of CD8+ T-cell responses against malaria liver stages

CD4+ T cells are crucial to the development of CD8+ T cell responses against hepatocytes infected with malaria parasites. In the absence of CD4+ T cells, CD8+ T cells initiate a seemingly normal differentiation and proliferation during the first few days after immunization. However, this response fails to develop further and is reduced by more than 90%, compared to that observed in the presence of CD4+ T cells. We report here that interleukin-4 (IL-4) secreted by CD4+ T cells is essential to the full development of this CD8+ T cell response. This is the first demonstration that IL-4 is a mediator of CD4/CD8 cross-talk leading to the development of immunity against an infectious pathogen.