In vitro stabilization and minimum active component of polygalacturonic acid synthase involved in pectin biosynthesis

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.     

An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins

We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.     

Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.     

Global advanced bioenergy potential under environmental protection policies and societal transformation measures

Bioenergy plays an important role in low greenhouse gas stabilization scenarios. Among various possible sources of bioenergy, dedicated bio‐crops could contribute to most of the potential. However, large scale bio‐crop deployment raises sustainability concerns. Policies to alleviate the pressure of bio‐crops on the terrestrial environment can affect bioenergy potential and production costs. Here, we estimated the maximum bioenergy potential under environmental protection policies (biodiversity and soil protection) and societal transformation measures from demand and supply side (demand‐side policy includes sustainable diet; supply‐side policy includes advanced technology and trade openness for food) by using an integrated assessment modelling framework, which consists of a general equilibrium model (Asian‐Pacific Integrated Model/Computable General Equilibrium) and a spatial land use allocation model (Asian‐Pacific Integrated Model/Platform for Land‐Use and Environmental Model). We found that the global advanced bioenergy potential under no policy was 245 EJ/year and that 192 EJ/year could be produced under US$5/GJ. These figures were 149 EJ/year and 110 EJ/year, respectively, under a full environmental policy. Biodiversity protection has a greater impact than soil protection due to its larger coverage and stronger implementation. Societal transformation measures effectively increase them to 186 EJ/year and 143 EJ/year, respectively, even under full environmental policies. These results imply that the large‐scale bioenergy deployment possibly needed for the climate target to limit the global mean temperature increase well below 2°C compared to the preindustrial level might face a trade‐off with environmental protection targets and that possible mitigation pathways in harmony with other environmental issues need to be explored.     

HLA genes and haplotypes in Ryukyuans suggest recent gene flow to the Okinawa Islands.

Polymorphism of HLA genes was investigated in a population sample of Ryukyuans living on the main island of Okinawa (n = 197), in the southwestern islands of Japan. Serological typing was applied to class I loci (HLA-A, -B, and -C) and to HLA-DRB1; nucleotide sequence-level typing was performed using PCR microtiter plate hybridization and PCR single-strand conformation polymorphism methods. Ryukyuans showed a higher frequency of DRB1*0405 and lower frequencies of DRB1*1502 and DRB1*1302 compared with Hondo Japanese living on main islands. Principal components and phylogenetic analyses of 12 East Asian populations, including Ryukyuans, were performed based on the allele frequencies of HLA-A, -B, and -DRB1. In the principal components analysis 3 Japanese populations (Ryukyuans, Hondo Japanese, and Ainu) formed a cluster and showed the highest affinity to 2 Korean populations. In the phylogenetic tree Ryukyuans and Ainu were neighbors, but the genetic distance between them was larger than the distances between Ryukyuans and Hondo Japanese and between Ryukyuans and Korean populations. The geographic cline of the predominant haplotype in Ryukyuans, A*24-B*54-DRB1*0405, suggests that an ancestral population possessing A*24-B*54-DRB1*0405 moved into the Okinawa Islands after the divergence of Ryukyuans from the Ainu. Such a recent gene flow, probably from South China to the Okinawa Islands, is considered the major cause of difference in genetic characteristics between Ryukyuans and the Ainu.     

A mixture of organisms affects cholesterol metabolism together with rat cecal flora.

The effects of a mixture of organisms on cecal fermentation and cholesterol metabolism in sham-operated and cecectomized rats were investigated. Male F344 rats, allocated into four groups: cecectomized rats fed a mixture of organisms (CEMO), cecectomized rats fed rice bran (CERB), sham-operated rats fed a mixture of organisms (SHMO), and sham-operated rats fed rice bran (SHRB) for 4 weeks. The diets had 0.5% cholesterol and 0.125% sodium cholate added. There were no significant differences in the body weight gain and food intake among the groups. The cecal pH in the SHMO group was significantly lower than that in the other groups. The total cholesterol and (VLDL + IDL + LDL)-cholesterol concentrations in serum were significantly lower in the SHMO group than that in the SHRB group, and the triacylglycerol concentration in the sham-operated rats tended to decrease compared to the cecectomized rats. The fecal cholesterol excretion in the CERB group was higher than that in the other groups, and that in the SHMO group was significantly higher than in the SHRB group. The acetic acid, propionic acid, n-butyric acid, and total short-chain fatty acid concentrations in the cecum contents were significantly higher in the SHMO group than those in the other groups. Streptococcus, Bifidobacterium, and Lactobacillus in the SHMO group tended to be higher than the other groups and Bacteroidaceae in the CEMO and CERB groups were significantly higher than that in the SHMO group. The results demonstrate that the mixture of organisms was fermented with the cecal contents and that the metabolites such as short-chain fatty acid lowered the serum total cholesterol and liver cholesterol concentrations in the rats fed a cholesterol-containing diet.     

Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis.

The purpose of this study was to investigate the mechanism of fatty acid-induced regulation of melanogenesis. An apparent regulatory effect on melanogenesis was observed when cultured B16F10 melanoma cells were incubated with fatty acids, i.e., linoleic acid (unsaturated, C18:2) decreased melanin synthesis while palmitic acid (saturated, C16:0) increased it. However, mRNA levels of the melanogenic enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2), were not altered. Regarding protein levels of these enzymes, the amount of tyrosinase was decreased by linoleic acid and increased by palmitic acid, whereas the amounts of TRP1 and TRP2 did not change after incubation with fatty acids. Pulse-chase assay by [35S]methionine metabolic labeling revealed that neither linoleic acid nor palmitic acid altered the synthesis of tyrosinase. Further, it was shown that linoleic acid accelerated, while palmitic acid decelerated, the proteolytic degradation of tyrosinase. These results suggest that modification of proteolytic degradation of tyrosinase is involved in regulatory effects of fatty acids on melanogenesis in cultured melanoma cells.     

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene. Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999