Bioinformatic and biochemical analysis of a novel maltose-forming α-amylase of the GH57 family in the hyperthermophilic archaeon Thermococcus sp. CL1.

Maltose-forming α-amylase is a glycoside hydrolase family 57 (GH57) member that is unique because it displays dual hydrolysis activity toward α-1,4- and α-1,6-glycosidic linkages and only recognizes maltose. This enzyme was previously identified only in Pyrococcus sp. ST04 (PSMA); however, we recently found two homologs subgroups in Thermococcus species. One subgroup (subgroup A) showed relatively high amino acid sequence similarity to PSMA (>71%), while the other subgroup (subgroup B) showed lower homology with PSMA (<59%). To characterize the subgroup B maltose-forming α-amylase from Thermococcus species (TCMA), we cloned the CL1_0868 gene from Thermococcus sp. CL1 and then successfully expressed the gene in Escherichia coli. Although TCMA has a different oligomeric state relative to PSMA, TCMA showed similar substrate specificity. However, TCMA was shown to hydrolyze maltooligosaccharides more easily than PSMA. Also, TCMA displayed different optimum conditions depending on the glycosidic linkage of the substrate. TCMA had the highest activity at 85°C and at pH 5.0 for α-1,4-glycosidic linkage hydrolysis whereas it showed its maximal activity to cleave α-1,6-glycosidic linkages at 98°C and pH 6.0.

Complete genome sequence of hyperthermophilic archaeon Thermococcus sp. ES1.

Thermococcus sp. strain ES1 is an anaerobic, hyperthermophilic archaeon from a hydrothermal vent that catabolizes sugars and peptides and produces H2S from S°, H2, acetate and CO2 as its primary metabolites. We present the complete genome sequence of this strain (1,957,742bp) with a focus on its substrate utilization and metabolite production capabilities. The sequence will contribute to the development of heterotrophic archaea for bioenergy production and biogeochemical modeling in hydrothermal environments.

Biotechnological production of arbutins (α- and ß-arbutins), skin-lightening agents, and their derivatives.

Arbutins (α- and ß-arbutins) are glycosylated hydroquinones that are commercially used in the cosmetic industry. These compounds have an inhibitory function against tyrosinase, a critical enzyme for generating pigments, which leads to the prevention of melanin formation, resulting in a whitening effect on the skin. Although ß-arbutin is found in various plants including bearberry, wheat, and pear, α-arbutin and other arbutin derivatives are synthesized by chemical and enzymatic methods. This article presents a mini-review of recent studies on the production of α-arbutin and other α- and ß-arbutin derivatives via enzymatic bioconversion methods. In addition, the structures of α- and ß-arbutin derivatives and their biological activities are discussed. The catalytic characteristics of various enzymes used in the biosynthesis of arbutin derivatives are also reviewed.

Micellar nanomedicine of human neuropeptide Y.

Human neuropeptide Y (NPY) is an important biologics that regulates a multitude of physiological functions and could be amenable to therapeutic manipulations in certain disease states. However, rapid (within minutes) enzymatic degradation and inactivation of NPY precludes its development as a drug. Accordingly, we determined whether self-association of NPY with biocompatible and biodegradable sterically stabilized phospholipid micelles (SSM) improves its stability and bioactivity. We found that in saline NPY spontaneously aggregates; however, in the presence of SSM it self-associates with the micelles as monomers. Three NPY molecules self-associate with 1 SSM at saturation. This process stabilizes the peptide in α-helix conformation, abrogates its degradation by dipeptidyl peptidase-4 and potentiates NPY-induced inhibition of cAMP elaboration in SK-N-MC cells. Collectively, these data indicate that self-association of NPY with SSM stabilizes and protects the peptide in active monomeric conformation, thereby amplifying its bioactivity in vitro. We propose further development of NPY in SSM as a novel, long-acting nanomedicine. FROM THE CLINICAL EDITOR: Human neuropeptide Y (NPY) regulates a multitude of physiological functions and could be amenable to therapeutic manipulations, which is currently limited by its short half life. Self-association of NPY with spherically stabilized micelles (SSM) protects and stabilizes the peptide in active monomeric conformation, thereby amplifying its bioactivity in vitro, enabling future therapeutic considerations.

The overmethylated genes in Helicobacter pylori-infected gastric mucosa are demethylated in gastric cancers.

BACKGROUND: The transitional-CpG sites between weakly methylated genes and densely methylated retroelements are overmethylated in the gastric mucosa infected with Helicobacter pylori (H. pylori) and they are undermethylated in the gastric cancers depending on the level of loss of heterozygosity (LOH) events. This study delineated the transitional-CpG methylation patterns of CpG-island-containing and -lacking genes in view of the retroelements. METHODS: The transitional-CpG sites of eight CpG-island-containing genes and six CpG-island-lacking genes were semi-quantitatively examined by performing radioisotope-labelling methylation-specific PCR under stringent conditions. The level of LOH in the gastric cancers was estimated using the 40 microsatellite markers on eight cancer-associated chromosomes. Each gene was scored as overmethylated or undermethylated based on an intermediate level of transitional-CpG methylation common in the H. pylori-negative gastric mucosa. RESULTS: The eight CpG-island genes examined were overmethylated depending on the proximity to the nearest retroelement in the H. pylori-positive gastric mucosa. The six CpG-island-lacking genes were similarly methylated in the H. pylori-positive and -negative gastric mucosa. In the gastric cancers, long transitional-CpG segments of the CpG-island genes distant from the retroelements remained overmethylated, whereas the overmethylation of short transitional-CpG segments close to the retroelements was not significant. Both the CpG-island-containing and -lacking genes tended to be decreasingly methylated in a LOH-level-dependent manner. CONCLUSIONS: The overmethylated genes under the influence of retroelement methylation in the H. pylori-infected stomach are demethylated in the gastric cancers influenced by LOH.

The gene-reduction effect of chromosomal losses detected in gastric cancers.

BACKGROUND: The level of loss of heterozygosity (LOH) that reduces a gene dose and exerts a cell-adverse effect is known to be a parameter for the genetic staging of gastric cancers. This study investigated if the cell-adverse effect induced with the gene reduction was a rate-limiting factor for the LOH events in two distinct histologic types of gastric cancers, the diffuse- and intestinal-types. METHODS: The pathologic specimens obtained from 145 gastric cancer patients were examined for the level of LOH using 40 microsatellite markers on eight cancer-associated chromosomes (3p, 4p, 5q, 8p, 9p, 13q, 17p and 18q). RESULTS: Most of the cancer-associated chromosomes were found to belong to the gene-poor chromosomes and to contain a few stomach-specific genes that were highly expressed. A baseline-level LOH involving one or no chromosome was frequent in diffuse-type gastric cancers. The chromosome 17 containing a relatively high density of genes was commonly lost in intestinal-type cancers but not in diffuse-type cancers. A high-level LOH involving four or more chromosomes tended to be frequent in the gastric cancers with intestinal and mixed differentiation. Disease relapse was common for gastric cancers with high-level LOH through both the hematogenous (38%) and non-hematogenous (36%) routes, and for the baseline-level LOH cases through the non-hematogenous route (67%). CONCLUSIONS: The cell-adverse effect of gene reduction is more tolerated in intestinal-type gastric cancers than in diffuse-type cancers, and the loss of high-dose genes is associated with hematogenous metastasis.

Nanomicellar paclitaxel increases cytotoxicity of multidrug resistant breast cancer cells.

Multidrug resistance (MDR) of breast cancer cells still represents an unmet medical need in chemotherapy. To this end, the purpose of this study was to determine efficacy of paclitaxel loaded in sterically stabilized, biocompatible and biodegradable sterically stabilized mixed phospholipid nanomicelles (SSMM; size, approximately 15 nm) and actively targeted vasoactive intestinal peptide-grafted SSMM (SSMM-VIP) in circumventing P-gp-mediated paclitaxel resistance in BC19/3 cells, a human breast cancer cell line that expresses >10-fold higher P-gp than its parental sensitive cell line, MCF-7. We found that in drug sensitive MCF-7 cells, paclitaxel loaded in SSMM (P-SSMM) and SSMM-VIP (P-SSMM-VIP) significantly inhibited cell growth in dose-dependent fashion (p<0.05). Both formulations were approximately 7-fold more potent than paclitaxel dissolved in DMSO (P-DMSO). Efficacy of P-SSMM and P-SSMM-VIP was similar (p>0.5). By contrast, in drug resistant BC19/3 cells, P-SSMM-VIP was significantly more effective than either P-SSMM or P-DMSO ( approximately 2- and 5-fold, respectively; p<0.05). Collectively, these data indicate that actively targeted paclitaxel-loaded SSMM-VIP overcomes multiple drug resistance of BC19/3 cells. We suggest this formulation should be further developed to treat MDR breast cancer.

Relationship between Psychological Factors and Quality of Life in Subtypes of Gastroesophageal Reflux Disease.

BACKGROUND/AIMS: Quality of life (QoL) is consistently decreased in gastroesophageal reflux disease (GERD), but the relationship between QoL and psychological factors in GERD has not yet been clearly defined. The present study investigated the relationship between the psychological factors of two subtypes of GERD and QoL. METHODS: A cohort of 769 participants underwent upper endoscopic evaluation in the health-promotion center of St. Paul's Hospital. The severity of GERD symptoms, psychological factors, and QoL were analyzed using the Visual Analogue Scale, the Hospital Anxiety and Depression Scale, and the abbreviated version of the World Health Organization Quality of Life instrument, respectively. RESULTS: Among the total of 769 participants, 153 participants were included in the exclusion criteria. Erosive reflux disease (ERD) and nonerosive reflux disease (NERD) were present in 106 (14%) and 61 (8%) of the participants, respectively, and 449 (58%) acted as controls. In each GERD group, the QoL had no correlatioion with the symptom severity. The scores for anxiety and depression were highest in the NERD group, and QoL scores were lower in both the ERD and NERD groups than in the control group. Anxiety and depression resulted in QoL scores being lower in both the ERD and NERD groups than in the nonanxiety and nondepressed groups, respectively. CONCLUSIONS: This study provides evidence that the QoL associated with the ERD and NERD subtypes may be more related to psychological factors than to symptom severity.

Targeted drug delivery crossing cytoplasmic membranes of intended cells via ligand-grafted sterically stabilized liposomes.

In this study, we tested whether sterically stabilized liposomes (SSL) with surface ligands specific for the mu opioid receptor (MOR) can actively target MOR-expressing cells. Dermorphin, a selective MOR agonist, was conjugated to DSPE-PEG(3400) to obtain DSPE-PEG(3400)-dermorphin. Dermorphin-grafted SSL (dermorphin-SSL) was prepared by thin-film rehydration-extrusion and post-insertion method. DSPE-PEG(3400)-dermorphin and dermorphin-SSL retained the affinity to MOR as determined by receptor binding assay using [(3)H]DAMGO, whereas plain SSL without surface ligands showed no binding to the receptor. Cellular uptake of cholesteryl BODIPY encapsulated dermorphin-SSL was studied by microplate spectrofluorometry as well as fluorescent and confocal microscopy. Significant fluorescence signal was observed inside CHO-hMOR cells after the treatment with dermorphin-SSL, indicative of MOR-mediated endocytosis. In contrast, no uptake of dermorphin-SSL was found in naive CHO cells or CHO-hDOR cells that lack MOR. Taken together, these results demonstrate that dermorphin-SSL delivery system is capable of targeting intracellular components of MOR-expressing cells. Such a system may be applied to carry pharmaceutical agents to achieve region-specific delivery of analgesics and/or to attenuate side effects associated with opioids.

Pluronic-grafted poly-(L)-lysine as a new synthetic gene carrier.

Genes are attractive candidates as therapeutic agents, and the development of safe and effective gene carriers is essential for the success of human gene therapy. To develop a gene delivery vector that shows low cytotoxicity and high efficiency, we synthesized poly-L-lysine-g-pluronic by conjugating poly-L-lysine (PLL) to pluronic, which is partially functionalized with para-nitrophenyl carbonate groups, and evaluated for its efficiency as a possible nonviral gene carrier candidate. Structural analysis of synthesized polymer was performed by using 1H-NMR. Gel retardation assay, zeta potential and size measurement confirmed that the new gene carrier made a compact complex with plasmid DNA. pCMV-beta-gal was used as a reporter gene, and the in vitro transfection efficiency was measured in HeLa cells by using the o-nitrophenyl-beta-D-galactopyranoside assay. The highest transfection efficiency among those tested was achieved at the 1:1 weight ratio of polymer:DNA, and a 3-fold increase in transfection efficiency was achieved by treatment of a lysosomotropic agent, chloroquine. Compared with unmodified PLL, PLL-g-pluronic showed about 2-fold increase in transfection efficiency with similar cytotoxicity specifically at the 1:1 weight ratio of polymer:DNA.