Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152–625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157–560 aa.) and PvRBP1a-C (606–962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.     

Comparison between entrapment methods for phenol removal and operation of bioreactor packed with co-entrapped activated carbon and Pseudomonas fluorescence KNU417

A microorganism capable of degrading phenol was isolated from crude oil contaminated soil and identified as Pseudomonas fluorescence. A porous polymer bead of polyvinyl alcohol (PVA) and Xanthan gum was found to be the best entrapment for phenol degradation in terms of bead shape (spherical form), bead strength, non-agglomeration, phenol degradation rate, and cell holding inside the bead. Activated carbon was co-immobilized with the microorganism in the bead, which readily adsorbed phenol to decrease initial phenol concentration. Due to the decreased phenol concentration, the cells needed shorter adaptation time after which the microorganism stably degraded phenol. When the bead containing microorganism with 1% of activated carbon was packed in a packed-bed bioreactor, the start-up period was shortened by 40 h and the removal efficiency of phenol during the period was increased by 28% than the case with only microorganism.     

The effects of sodium bisulfite in extraction of PHB by hypochlorite

Summary Molecular weight of PHB becomes low when using a hypochlorite extraction method for PHB separation. This disadvantage was overcome by adding sodium bisulfite which is an anti-oxidant. The molecular weight drop was decreased from 30 % to 14 % by adding sodium bisulfite. It was also found that raising the pH to 12 and the addition of a surfactant resulted in the improvement of the PHB purity.     

Conversion of cell-survival activity of Akt into apoptotic death of cancer cells by two mutations on the BIM BH3 domain

Survival and proliferation of cancer cells are often associated with hyperactivity of the serine/threonine kinase, Akt. Herein, we show that prosurvival activity of Akt can be converted into prodeath activity by embedding an Akt recognition sequence in the apoptogenic BH3 domain of human BIM. The recognition sequence was created by introducing two mutations, I155R and E158S, into the core region of the BIM BH3 domain. Although a 21-mer BIM BH3 peptide containing these two mutations bound weakly to BCL-X_L and BCL-2, this peptide with phosphorylation of Ser158 bound to these proteins with a dissociation constant of <10 nM. The crystal structure of the phosphorylated peptide bound to BCL-X_L revealed that the phospho-Ser158 makes favorable interactions with two BCL-X_L residues, which cannot be formed with unphosphorylated Ser158. Remarkably, the designed peptide showed a cytotoxic effect on PTEN-null PC3 tumor cells whose Akt activity is aberrantly high. The cell-killing activity disappeared when the cellular Akt activity was lowered by ectopic PTEN expression. Thus, these results lay a foundation for developing a peptide or protein agent that is dormant in normal cells but is transformed into a potent apoptogenic molecule upon phosphorylation by hyperactivity of Akt in cancer cells.The interplay between the BCL-2 family proteins regulates mitochondrion-mediated apoptotic cell death.^{1, 2} The BCL-2 family proteins are characterized by having at least one BCL-2 homology (BH) domain, and they are classified into three distinct subgroups based on their functional and structural features. One subgroup consists of BAX and BAK, which contain the BH1-BH4 domains and mediate apoptosis by increasing the permeability of the mitochondrial outer membrane (MOM) and thus leading to the release of the apoptogenic factors, such as cytochrome c and Smac/Diablo.^{3, 4, 5, 6} Another subgroup is composed of antiapoptotic proteins, BCL-2, BCL-X_L, BCl-w, MCL-1, A1 and BCL-B, which contain the BH1-BH4 domains that are arranged to form an extended hydrophobic groove known as the BH3-binding groove.⁷ The remaining subgroup is composed of a diverse set of proteins that are unrelated to each other except for the possession of the BH3 domain.⁷ These BH3-only proteins sense and convey apoptotic cell death signals, ultimately leading to the activation of BAX and BAK.^{8, 9} The antiapoptotic BCL-2 subfamily proteins bind the BH3 domain of BAX/BAK and of the BH3-only proteins through their BH3-binding groove.^{10, 11, 12, 13, 14, 15}Biochemical studies have discovered that a number of the BH3-only proteins termed ‘activators'', such as BID and BIM, bind directly to BAX and induce its activation, whereas other BH3-only proteins termed ‘sensitizers'' induce apoptosis by releasing the activators sequestered by the antiapoptotic proteins.^{5, 16, 17} A recent crystallographic study revealed that the BID BH3 peptide binds to the canonical BH3-binding groove of BAX and induces a pronounced conformational change that exposes the BH3 domain of BAX.¹⁸ The activated BAX oligomerizes to induce the permeabilization of the MOM.⁶ The antiapoptotic BCL-2 proteins were suggested to sequester the BH3 domains of both BAX and the activator BH3-only proteins to prevent the BAX oligomerization.¹⁸Apoptosis is attenuated in cancer cells because of the abundance of antiapoptotic BCL-2 proteins and/or prevention of apoptosis induction. Anticancer BH3 peptides have been developed, especially those derived from BIM, which interacts with all of the antiapoptotic proteins with extremely high affinity.^{15, 19} These BH3 peptides exhibit a broad and multimodal targeting of the BCL-2 family proteins.^{20, 21, 22} Promising small molecular anticancer compounds have also been developed that mimic the BH3 peptides and bind to the surface groove of the antiapoptotic proteins.²³ ABT-737 and ABT-263 selectively bind to and lower the amounts of the functional BCL-2, BCL-X_L and BCL-w proteins to induce the apoptotic death of tumor cells that depend especially on the overexpression of the three proteins.^{24, 25} The BH3 peptides and the BH3 mimetics both bear an intrinsic shortcoming in that they inhibit the BCL-2 family proteins not only in cancer cells but also in normal cells as they cannot distinguish cancerous from normal cells.One of the hallmarks of many cancer and tumor cells is the hyperactivation of the serine/threonine (Ser/Thr) protein kinase Akt, which is a key signaling molecule in the cellular survival pathway.²⁶ In many types of cancers, including glioma, prostate cancer and breast cancer, Akt is required to maintain a proliferative state and for progression into a more malignant state in conjunction with genetic mutations.^{26, 27, 28}We set out to develop a molecule that can respond to the hyperactivity of Akt and can lead to the death of cancer cells. Herein, we describe the embedment of the Akt recognition sequence into the BIM BH3 peptide and the cancer cell-specific apoptogenic property of the resulting BIM BH3 peptide variant characterized by X-ray crystallography, calorimetry and cell-based biochemistry.     

l-Ribulose production from l-arabinose by an l-arabinose isomerase mutant from Geobacillus thermodenitrificans

Geobacillus thermodenitrificans, with a double-site mutation in L: -arabinose isomerase, produced 95 g L-: ribulose l(-1 ) from 500 g L: -arabinose l(-1) under optimum conditions of pH 8, 70 degrees C, and 10 units enzyme ml(-1) with a conversion yield of 19% over 2 h. The half-lives of the mutated enzyme at 70 and 75 degrees C were 35 and 4.5 h, respectively.     

Optimization of a Novel Two-step Process Comprising Re-esterification and Transesterification in a Single Reactor for Biodiesel Production Using Waste Cooking Oil

Waste cooking oil (WCO) has attracted attention as a non-edible feedstock for biodiesel. Although an alkali catalyst has several advantages over an acid catalyst in biodiesel production, biodiesel conversion from WCO is only 5.2% when using an alkali catalyst (NaOH), owing to its high free fatty acid (FFA) content of 4.2%. In this study, a novel two-step process in a single reactor, comprised of re-esterification of the FFAs with crude glycerol, using a Tin (II) chloride (SnCl₂) catalyst, and subsequent transesterification with methanol, using an alkali catalyst, was adopted, and each step was optimized. This study revealed that the FFA content after re-esterification should be approximately 1.5%, not only to save glycerol and the catalyst involved in the re-esterification, but also to achieve high biodiesel conversion during the transesterification. An alkaline catalyst was successfully used to produce biodiesel in the second step, and a 92.8% conversion to biodiesel was achieved under the optimized conditions (0.6% catalyst relative to WCO, 0.2mL-methanol/WCO, 70ºC, 3 h). Overall, this novel two-step process achieved highly enhanced biodiesel conversion (4.0% to 92.8%) with significantly reduced reaction time (12 h to 4 h) and methanol requirements (15 mL/g-WCO to 0.2 mL/g-WCO).     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence imaging to assess early therapeutic response to tumor progression in a xenograft cancer model

Bioimaging technology plays an important role in assessment of therapeutic response in cancer therapy. Here, we report a non-invasive monitoring system for measuring tumor growth by in vivo fluorescence imaging. Target cells for xenograft tumor induction were manipulated by cell-surface fluorescence labelling. Fluorescence was clearly detected in vitro and in vivo without affecting cytotoxicity. Anti-tumor efficacy was evaluated by directly measuring the fluorescence signal of a progressive tumor in a xenograft model. This non-invasive in vivo monitoring system can be used to assess the early response to antitumor therapeutics and may be a valuable tool to replace or complement traditional caliper-based methods for preclinical studies.     

Effects of deer,mice and dwarf bamboo on the emergence,survival and growth of <Emphasis Type="Italic">Abies homolepis</Emphasis> (Piceaceae) seedlings

In the subalpine mixed forest of Mt. Ôdaigahara, mid-western Japan, the understory is dominated by dwarf bamboo (Sasa nipponica), which is the major forage of overly populous sika deer (Cervus nippon). In the present study, we monitored the survival and growth of Abies homolepis seedlings over 5years to determine how they responded to the experimental removal of dwarf bamboo and to the exclusion of sika deer and mice (Apodemus argenteus and A.speciosus). Deer and dwarf bamboo reduced the survival of seedlings but had different effects on growth. The stems of seedlings were shorter in the presence of deer, indicating that taller seedlings were apt to be browsed by deer, whereas the diameters of seedlings were smaller in the presence of dwarf bamboo, mainly owing to its shading effect. The presence of mice decreased the number of seedlings germinating in a particular site, but had no effect on seedling survival after germination. There was no significant indirect effect whereby the survival of seedlings was predicted to be facilitated by the decreased biomass of bamboo because of grazing by deer. We supposed that this might be because the direct negative effect of deer was so large as to conceal the positive indirect effect.