Pisidium coreanum Inhibits Multinucleated Osteoclast Formation and Prevents Estrogen-Deficient Osteoporosis.

Mollusks have served as important sources of human food and medicine for a long time. Raw Pisidium coreanum, a freshwater bivalve of the phylum Mollusca, is used in traditional therapies in parts of Asia. However, the therapeutic effects of Pisidium coreanum on bone diseases are not known. We investigated the functional roles of Pisidium coreanum in osteoporotic bone diseases. Pisidium coreanum inhibited the differentiation of bone marrow-derived monocytic cells into mature osteoclasts in vitro. The ovariectomized mice that received oral administration of Pisidium coreanum showed improvements in both trabecular and cortical bones. This preventive activity of Pisidium coreanum against bone loss was due to limited osteoclast maturation with reduced osteoclast surface extent in trabecular bone tissue. The formation of large multinucleated osteoclasts in vitro was significantly decreased in response to Pisidium coreanum, consistent with the reduced expression levels of osteoclast markers and fusion-related genes, such as NFATc1, p65, integrin αvß3, DC-STAMP, OC-STAMP, Atp6v0d2, FAK, CD44, and MFR. These data suggest that Pisidium coreanum inhibits osteoclast differentiation by negatively regulating the fusion of mononuclear osteoclast precursors. Thus, our data demonstrate the ability of Pisidium coreanum to effectively prevent estrogen-deficient osteoporosis through inhibition of multinucleated osteoclast formation.

Calcium Involved Directional Organization of Polymer Chains in Polyester Nanogranules in Bacterial Cells.

Soil bacteria accumulate polyesters (typically poly([R]-3-hydroxybutyrate (PHB), in which one end of the chain terminates with a carboxyl group) in the form of hydrated, amorphous nanogranules in cells. However, it is not clear what drives the structure of these biomaterials inside bacterial cells. Here, we determined that calcium guides intracellular formation of PHB nanogranules. Our systematic study using the surface zeta potential measurement and the carboxyl-specific SYTO-62 dye binding assay showed that the terminal carboxyl is not exposed to the granule surface but is buried inside native "unit-granules" comprising the mature granule. Extracellular Ca2+ was found to mediate the formation of these PHB unit-granules, with uptaken Ca2+ stored inside the granules. Comparative [Ca2+]-dependent fluorescence spectroscopy revealed that the native granules in Cupriavidus necator H16 act as a Ca2+ storage system, presumably for the regulation of its cytosolic Ca2+ level, but those from recombinant Escherichia coli do not. This study reveals intimate links between Ca2+ and native granule formation, and establishes a novel mechanism that intracellular PHB granules function as Ca2+ storage in order to relieve soil bacteria from Ca2+ stress.

Roles of Mitogen-Activated Protein Kinases in Osteoclast Biology.

Bone undergoes continuous remodeling, which is homeostatically regulated by concerted communication between bone-forming osteoblasts and bone-degrading osteoclasts. Multinucleated giant osteoclasts are the only specialized cells that degrade or resorb the organic and inorganic bone components. They secrete proteases (e.g., cathepsin K) that degrade the organic collagenous matrix and establish localized acidosis at the bone-resorbing site through proton-pumping to facilitate the dissolution of inorganic mineral. Osteoporosis, the most common bone disease, is caused by excessive bone resorption, highlighting the crucial role of osteoclasts in intact bone remodeling. Signaling mediated by mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, has been recognized to be critical for normal osteoclast differentiation and activation. Various exogenous (e.g., toll-like receptor agonists) and endogenous (e.g., growth factors and inflammatory cytokines) stimuli contribute to determining whether MAPKs positively or negatively regulate osteoclast adhesion, migration, fusion and survival, and osteoclastic bone resorption. In this review, we delineate the unique roles of MAPKs in osteoclast metabolism and provide an overview of the upstream regulators that activate or inhibit MAPKs and their downstream targets. Furthermore, we discuss the current knowledge about the differential kinetics of ERK, JNK, and p38, and the crosstalk between MAPKs in osteoclast metabolism.

A Lipid Emulsion Reverses Toxic-Dose Bupivacaine-Induced Vasodilation during Tyrosine Phosphorylation-Evoked Contraction in Isolated Rat Aortae.

The goal of this in vitro study was to examine the effect of a lipid emulsion on toxic-dose bupivacaine-induced vasodilation in a model of tyrosine phosphatase inhibitor sodium orthovanadate-induced contraction in endothelium-denuded rat aortae and to elucidate the associated cellular mechanism. The effect of a lipid emulsion on vasodilation induced by a toxic dose of a local anesthetic during sodium orthovanadate-induced contraction was examined. In addition, the effects of various inhibitors, either bupivacaine alone or a lipid emulsion plus bupivacaine, on protein kinase phosphorylation induced by sodium orthovanadate in rat aortic vascular smooth muscle cells was examined. A lipid emulsion reversed the vasodilation induced by bupivacaine during sodium orthovanadate-induced contraction. The lipid emulsion attenuated the bupivacaine-mediated inhibition of the sodium orthovanadate-induced phosphorylation of protein tyrosine, c-Jun NH2-terminal kinase (JNK), myosin phosphatase target subunit 1 (MYPT1), phospholipase C (PLC) γ-1 and extracellular signal-regulated kinase (ERK). These results suggest that a lipid emulsion reverses toxic-dose bupivacaine-induced vasodilation during sodium orthovanadate-induced contraction via the activation of a pathway involving either tyrosine kinase, JNK, Rho-kinase and MYPT1 or tyrosine kinase, PLC γ-1 and ERK, and this reversal is associated with the lipid solubility of the local anesthetic and the induction of calcium sensitization.

Lipid Emulsion Inhibits Apoptosis Induced by a Toxic Dose of Verapamil via the Delta-Opioid Receptor in H9c2 Rat Cardiomyoblasts.

The goals of this study were to investigate the effects of lipid emulsion (LE) on apoptosis induced by a toxic dose of verapamil in H9c2 cells and to elucidate the associated cellular mechanism. The effects of LE alone and combined with an inhibitor on the decreases in cell counts and viability induced by verapamil and diltiazem were examined using the MTT assay. The effects of verapamil alone, combined LE and verapamil treatment, and combined inhibitor, LE and verapamil treatment on cleaved caspase-3, caspase-8 and Bax expression, were examined using Western blotting. The effects of verapamil alone and combined with LE on the number of TUNEL-positive H9c2 cells were also examined. LE attenuated the decreases in cell counts and viability induced by verapamil and diltiazem. However, the magnitude of the LE-mediated attenuation of decreased cell viability was enhanced by verapamil compared with diltiazem treatment. Naloxone, naltrindole hydrochloride, LY294002 and MK-2206 inhibited the LE-mediated attenuation of increased cleaved caspase-3 and caspase-8 expression induced by verapamil. LE attenuated the increase in the number of TUNEL-positive cell induced by verapamil. These results suggest that LE attenuates apoptosis induced by verapamil via activation of the delta-opioid receptor, phosphoinositide 3-kinase and Akt.

Nanoscale poly(4-hydroxybutyrate)-mPEG carriers for anticancer drugs delivery.

Despite several advancements in chemotherapy, cancer is still the second most frequent cause of mortality worldwide. Drug delivery to solid tumors is one of the most challenging aspects in cancer therapy. In pharmaceutical industries biodegradable polymeric nanoparticles as drug carriers have attracted great research interest because of their biocompatibility, biodegradability and sustained release of drugs. In our study we prepared poly(4-hydroxybutyrate)-mPEG (P(4HB)-mPEG) nanocarriers for the delivery of cisplatin as anticancer drug to mouse hippocampal HT22 cells. P(4HB) is more suitable candidate to be utilized in pharmaceutical industries due to its wide medical applications. P(4HB) is a homopolymer of 4-hydroxybutyrate (4HB), and belongs to a diverse class of materials called polyhydroxyalkanoates (PHA) produced by microorganisms inside the cells as energy storage materials. P(4HB) has certain unique properties such as biocompatibility and rapid in vivo degradation, which differentiate it from others PHA based polymers. Novel amorphous amphiphilic block copolymer P(4HB)-mPEG nanocarriers were prepared and characterized. Flow cytometry, and confocal microscopy revealed a suppression effect by the cisplatin loaded nanocarriers on HT22 cell growth, and enhancement of apoptotic process of the cells compared to free drug treated cells. The amorphous polymeric nanocarriers could be effective vehicles for the sustained delivery of toxic anticancer drugs for the therapy of cancer.

Simultaneous inhibition of rhamnolipid and polyhydroxyalkanoic acid synthesis and biofilm formation in Pseudomonas aeruginosa by 2-bromoalkanoic acids: effect of inhibitor alkyl-chain-length.

Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and polyhydroxyalkanoic acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty acids of 2-bromohexanoic acid (2-BrHA), 2-bromooctanoic acid (2-BrOA) and 2-bromodecanoic acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

Amorphous amphiphilic P(3HV-co-4HB)-b-mPEG block copolymer synthesized from bacterial copolyester via melt transesterification: nanoparticle preparation, cisplatin-loading for cancer therapy and in vitro evaluation.

Cisplatin is a chemotherapeutic agent used against a variety of tumors. We determined the efficacy and bioavailability of cisplatin in the form of cisplatin-loaded self-assembled amphiphilic copolymer nanoparticles (NPs). Non-crystallizing bacterial copolyester was employed as hydrophobic segment to increase drug loading efficiency. Novel amorphous amphiphilic block copolymer P(3HV-co-4HB)-b-mPEG was synthesized from bacterial copolyester poly(3-hydroxyvalerate-co-4-hydroxybutyrate) coupled via transesterification reaction using bis(2-ethylhexanoate) tin catalyst to monomethoxypoly(ethylene glycol). The product was characterized, and core-shell particles with nanometer size range were prepared by emulsification-solvent evaporation method. Transmission electron microscopy (TEM) examination revealed that the NPs took the shape of spheres with inner concealed core of hydrophobic P(3HV-co-4HB) polymer and the outer shell formed by hydrophilic mPEG segment. The in vitro release profile of cisplatin from the core hydrophobic domain showed a sustained release of the drug. TEM and confocal microscopy examination revealed clearly the internalization of cisplatin-loaded NPs into the tumor cells. MTT assay, flow cytometry, western blot and confocal microscopy revealed a suppression effect by the NPs on tumor cell growth, and enhancement of apoptotic process of the tumor cells compared to free drug treated cells. The amorphous polymeric NPs could be effective vehicles for the sustained delivery of toxic anticancer drugs.

Synthesis and characterization of PHV-block-mPEG diblock copolymer and its formation of amphiphilic nanoparticles for drug delivery.

Despite the recent research interest in the field of nanoparticles delivery system, their structure modification and transport behavior of various hydrophobic drugs is poorly developed. In this article the synthesis of novel amphiphilic diblock copolymer poly([R]-3-hydroxyvalerate)-block-monomethoxy poly(ethylene glycol) (PHV-block-mPEG) was undertaken by modifying the structure of biodegradable and hydrophobic poly([R]-3-hydroxyvalerate) (PHV) with hydrophilic monomethoxy poly(ethylene glycol) (mPEG). The chemical combination of the two blocks was carried out in the melt using bis(2-ethylhexanoate) tin as transesterification catalyst. The synthesized product was characterized by gel permeation chromatography (GPC), 1H nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) analysis. The block copolymer self-assembled into amphiphilic nanoparticles with a core of hydrophobic PHV and a shell of hydrophilic mPEG in aqueous solution. Characterization of the nanoparticles showed the formation of discrete, spherically shaped nanoparticles with mean particle size of 200 +/- 1 nm and zeta potential of -14 +/- 1 mV. A hydrophobic drug thymoquinone was efficiently incorporated into the core hydrophobic domain of the nanoparticles and its release kinetics was studied in vitro. The amphiphilic PEGylated nanoparticles showed biocompatibility when checked in neuronal hippocampal cells of prenatal rat. Our results suggest that the amphiphilic nanoparticles with core-shell structures are potentially useful to develop novel drug carriers.

Metabolic relationship between polyhydroxyalkanoic acid and rhamnolipid synthesis in Pseudomonas aeruginosa: comparative ¹³C NMR analysis of the products in wild-type and mutants.

Polyhydroxyalkanoic acids (PHAs) and rhamnolipids considered as biotechnologically important compounds are simultaneously produced by Pseudomonas aeruginosa. Both are synthesized from common precursors, (R)-3-hydroxyfatty acids. To find the probable metabolic relationship between their syntheses, we investigated the PHA and rhamnolipids production in four pha (phaC1, phaC2, phaZ, and phaG), four rhl (rhlA, rhlB, rhlR, and rhlI) and rpoS mutant strains of P. aeruginosa PA14 and PAO1 grown in minimal medium containing 70 mM fructose or 30 mM decanoic acid. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxyfatty acid precursors become more available for PHA synthesis when rhamnolipids synthesis is absent. However, compared to the wild-type strains, rhamnolipids production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipids production in P. aeruginosa could be tightly regulated at the transcriptional level by a quorum-sensing response. The metabolic pathways for PHA and rhamnolipid synthesis from medium-chain-length fatty acids were also investigated using octanoic-1-¹³C acid. ¹³C NMR analysis revealed that the monomer-unit (R)-3-hydroxyoctanoate-1-¹³C being converted from the octanoic acid substrate was effectively incorporated into PHA. In the rhamnolipid synthesis, the (R)-3-hydroxyoctanoate-1-¹³C is suggested to be firstly converted to (R)-3-hydroxydecanoate-1,3-¹³C via fatty acid de novo biosynthesis pathway and then further processed into (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic acids (HAAs) via RhlA. The ratio of mono- to dirhamnolipids in the product depended on the type of carbon sources. The rhlB mutant could be exploited as an efficient producer of the important biosurfactant HAAs (e.g., ~700 mg/L HAAs was obtained when grown on 60 mM octanoic acid).

Amphiphilic PHA-mPEG copolymeric nanocontainers for drug delivery: preparation, characterization and in vitro evaluation.

Amphiphilic biodegradable core-shell nanoparticles were prepared by emulsification-solvent evaporation technique from diblock copolymers which were synthesized by chemical coupling of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) to monomethoxy poly(ethylene glycol) (mPEG) through transesterification reaction. The nanoparticles were found to be assembled in aqueous solution into an outer hydrophilic shell of mPEG connected to the interior hydrophobic polyhydroxyalkanoate (PHA) copolymer core, which was identified by a comparative analysis of enzymatic degradation of the mPEG-coupled and non-coupled PHA nanoparticles. Morphological examination under atomic force microscope showed the formation of smooth spherically shaped nanoparticles. The average particle sizes and zeta potentials of amphiphilic nanoparticles were in the range of 112-162 nm and -18 to -27 mV, respectively. A hydrophobic drug thymoquinone was encapsulated in the nanoparticles and its release kinetics was studied. The in vitro cytotoxicity evaluation of the nanoparticles on prenatal rat neuronal hippocampal and fibroblast cells revealed that biocompatibility of the amphiphilic nanoparticles was generally independent of the ratio of comonomer units in the PHA block. In conclusion, the amphiphilic nanoparticles contained the hydrophobic PHA segments buried in the core and could thus be used as safe carriers for the controlled release of variety of hydrophobic drugs.

Production of poly-N-acetylglucosamine by Staphylococcus saprophyticus BMSZ711: characterization and production optimization.

This is the first report on the characterization and production optimization of poly-N-acetylglucosamine (PNAG) in Staphylococcus saprophyticus. A strain producing glucosamine exopolysaccharide was isolated and characterized by biochemical test and 16S rRNA gene sequence homology analysis and named as S. saprophyticus BMSZ711. The molecular mass of the purified exopolymer was about 12 kDa. Digestion of the PNAG with DispersinB proved that it has beta-1,6 linkage. BMSZ711 can only produce PNAG when grown in M1 minimal medium but not in nutrient rich medium with optimum temperature of 30 degrees C and pH of 7. Glycerol and ammonium sulfate were found to be the best carbon and nitrogen source, respectively. Maximum PNAG production was obtained when glycerol 100mM, ammonium sulfate 0.3%, yeast extract 1.5 g/L, sodium chloride 10 g/L and valine 2mM were used.

Isolation and characterization of a transposon mutant of Pseudomonas fluorescens BM07 enhancing the production of polyhydroxyalkanoic acid but deficient in cold-induced exobiopolymer production.

Pseudomonas fluorescens BM07 is known to produce cold-induced exobiopolymer, which is mainly composed of water-insoluble hydrophobic polypeptides (up to 85%) and saccharides (8%), by decreasing the culture temperature down to as low as 10 degrees C. We screened for transposon insertion mutants of P. fluorescens BM07 that were unable to produce the exobiopolymer. Among the eight mutants that showed the deficiency of exobiopolymer and O-lipopolysaccharide, one mutant BM07-59 that had the highest polyhydroxyalkanoates (PHA) production was selected. The transposon inserted gene in BM07-59 was identified as galU. The disruption of the gene galU coded for the putative product, UDP-glucose pyrophosphorylase (GalU), resulted in 1.5-fold more accumulation of PHA compared with the wild-type strain from 70 mM fructose or galactose at 30 degrees C. Electrophoretic analysis of lipopolysaccharide showed that the mutant lacked the O-antigen lipopolysaccharide bands. The glycosyl composition of the lipopolysaccharide produced by the mutant strain was significantly different from that of the wild-type strain. We suggest that the deletion of galU could be a way to shift carbon flux efficiently from exobiopolymer toward PHA in P. fluorescens BM07.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with omega-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid beta-oxidation inhibition.

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07.

Correlating metal ionic characteristics with biosorption capacity of Staphylococcus saprophyticus BMSZ711 using QICAR model.

Quantitative Ion Character-Activity Relationship (QICAR) was used for correlating metal ionic properties with maximum biosorption capacity (q(max)). Heat inactivated biomass of Staphylococcus saprophyticus BMSZ711 was studied for biosorption of nine metal ions. Influence of contact time and initial pH was checked. q(max) was determined by Langmuir isotherm and followed a descending sequence (in mmol/g): Pb(2+)>Cd(2+)>Cr(3+)>Zn(2+)>Hg(2+)>Cu(2+)>Co(2+)>Ni(2+)>K(+). q(max) values was modeled with 20 metal ionic characteristics, among these covalent index (X(m)(2)r) was best fitted with q(max) for all metal ions tested, in the following model: q(max)=0.09+0.11(X(m)(2)r) (R(2)=0.73, AIC=-4.14). Classification of metal ions according to valence or soft/hard improved QICARs modeling and more characteristics significantly correlated with q(max) which revealed that covalent bonding played major role in biosorption of soft metal ions and ionic bonding for borderline and hard ions. Biosorption capacity was most effectively predicted (R(2)=0.99, AIC=-8.04) with a two variable model containing electro-negativity (X(m)) and softness index (o(rho)(')).

Shifting of the distribution of aromatic monomer-units in polyhydroxyalkanoic acid to longer units by salicylic acid in Pseudomonas fluorescens BM07 grown with mixtures of fructose and 11-phenoxyundecanoic acid.

Medium-chain-length-polyhydroxyalkanoic acids (MCL-PHAs) formed in Pseudomonas spp. have a rather broad distribution of monomer-units whose precursors are supplied via beta-oxidation degradation of MCL fatty acids fed as the carbon source and/or via PhaG enzyme catalyzing the acyl-group transfer from 3-hydroxyacyl-ACPs derived from acetyl-CoA to coenzyme A. It was found that salicylic acid (SA), in a concentration dependent manner, suppressed the accumulation of PHA in Pseudomonas fluorescens BM07 from fructose as well as shifted the distribution of monomer-units derived from a MCL fatty acid co-added as carbon source (e.g., 11-phenoxyundecanoic acid (11-POU)) to longer monomer-units. Both SA and acrylic acid were found to induce high accumulations of 3-ketohexanoic acid in BM07 wild-type cells grown with n-hexanoic acid as well as to inhibit the formation of acetyl-CoA from acetoacetyl-CoA by BM07 cell extract, suggesting that 3-ketoacyl-CoA thiolase is their common beta-oxidation target. The structural motif of acrylic acid present in the molecular structure of SA may self-explain the similar actions of the two inhibitors. A comparison of monomer modulation between BM07 wild-type and DeltaphaG mutant cells grown on the mixtures of fructose and 11-POU revealed that both PhaG and beta-oxidation inhibitor may play a critical role in the synthesis of PHA with longer side-chain omega-functional substitutions.

Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2.

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of PHAMCL from waste vegetable oil. The proportion of 3- hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.

Enhanced biosorption of mercury(II) and cadmium(II) by cold-induced hydrophobic exobiopolymer secreted from the psychrotroph Pseudomonas fluorescens BM07.

The cells of psychrotrophic Pseudomonas fluorescens BM07 were found to secrete large amounts of exobiopolymer (EBP) composed of mainly hydrophobic (water insoluble) polypeptide(s) (as contain approximately 50 mol% hydrophobic amino acids, lacking cysteine residue) when grown on fructose containing limited M1 medium at the temperatures as low as 0-10 degrees C but trace amount at high (30 degrees C, optimum growth) temperature. Two types of nonliving BM07 cells (i.e., cells grown at 30 degrees C and 10 degrees C) as well as the freeze-dried EBP were compared for biosorption of mercury (Hg(II)) and cadmium (Cd(II)). The optimum adsorption pH was found 7 for Hg(II) but 6 for Cd(II), irrespective of the type of biomass. Equilibrium adsorption data well fitted the Langmuir adsorption model. The maximum adsorption (Q(max)) was 72.3, 97.4, and 286.2 mg Hg(II)/g dry biomass and 18.9, 27.0, and 61.5 mg Cd(II)/g dry biomass for cells grown at 30 degrees C and 10 degrees C and EBP, respectively, indicating major contribution of heavy metal adsorption by cold-induced EBP. Mercury(II) binding induced a significant shift of infrared (IR) amide I and II absorption of EBP whereas cadmium(II) binding showed only a very little shift. These IR shifts demonstrate that mercury(II) and cadmium(II) might have different binding sites in EBP, which was supported by X-ray diffraction and differential scanning calorimetric analysis and sorption results of chemically modified biomasses. This study implies that the psychrotrophs like BM07 strain may play an important role in the bioremediation of heavy metals in the temperate regions especially in the inactive cold season.

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis.

OBJECTIVE: To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. DESIGN: Prospective model study. SETTING: Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. ANIMAL(S): F1 hybrid B6D2F1 mice. INTERVENTION(S): Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. MAIN OUTCOME MEASURE(S): Preimplantation development and stem cell characterization. RESULT(S): More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. CONCLUSION(S): Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

Swinging effect of salicylic acid on the accumulation of polyhydroxyalkanoic acid (PHA) in Pseudomonas aeruginosa BM114 synthesizing both MCLandSCL-PHA.

A bacterium, Pseudomonas aeruginosa BM114, capable of accumulating a blend of medium-chain-length (MCL)- and short-chain-length (SCL)-polyhydroxyalkanoic acid (PHA), was isolated. Salicylic acid (SA), without being metabolized, was found to specifically inhibit only the accumulation of MCL-PHA without affecting cell growth. An addition of 20 mM SA selectively inhibited the accumulation of MCL-PHA in decanoate-grown cells by 83% of the control content in one-step cultivation, where overall PHA accumulation was inhibited by only approximately11%. Typically, the molar monomerunit ratio of the PHA for 25 mM decanoate-grown cells changed from 46:4:25:25 (=[3-hydroxybutyrate]:[3-hydroxycaproate]: [3-hydroxyoctanoate]:[3-hydroxydecanoate]) at 0 mM SA (dry cell wt, 1.97 g/l; PHA content, 48.6 wt%) to 91:1:4:4 at 20 mM SA (dry cell wt, 1.85 g/l; PHA content, 43.2 wt%). Thus, the stimulation of SCL-PHA accumulation was observed. Growth of P. aeruginosa BM114 on undecanoic acid also produced a PHA blend composed of 47.4% P(3HB-co-3- hydroxyvalerate) and 52.6% P(3-hydroxyheptanoate-co-3- hydroxynonanoate-co-3-hydroxyundecanoate). Similar to the case of even-carboxylic acids, SA inhibited the accumulation of only MCL-PHA, but stimulated the accumulation of SCLPHA. For all medium-chain fatty acids tested, SA induced a stimulation of SCL-PHA accumulation in the BM114 strain. SA could thus be used to suppress only the formation of MCL-PHA in Pseudomonas spp. accumulating a blend of SCL-PHA and MCL-PHA.