The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters

Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.     

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.     

Oxidative Damage to Membranes by a Combination of Aluminum and Iron in Suspension-Cultured Tobacco Cells

Aluminum (Al) and ferrous iron [Fe(II)] are separately non-toxicto cultured tobacco cells in nutrient solution. However, Aland Fe(II) together cause the peroxidation of membrane iipids,the accumulation of Al and Fe, and the loss of viability [Onoet al. (1995) Plant Cell Physiol. 36: 115]. We investigatedthe cause-and-effect relationships of these various responses.In cells exposed to Fe(TT) or Fe(III)-EDTA, both the peroxidationof ipids and the loss of viability were similarly enhanced byAlCl₃ in a dose-dependent manner. During exposure to AlCl₃,the accumulation of Al and the loss of viability became apparentrapidly and simultaneously at 8 h, whereas both the peroxidationof lipids and the accumulation of Fe occurred at later times.However, lipophilic antioxidants protected cells efficientlynot only from the peroxidation of Iipids but also from the lossof viability and the accumulation of Al and Fe. These resultssuggest that the peroxidation of Iipids in the plasma membranethat is caused by both Al and Fe leads to the accumulation ofAl and Fe and the loss of viability. (Received April 18, 1997; Accepted October 1, 1997)     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Cascading effects of larval Crucian carp introduction on phytoplankton and microbial communities in a paddy field: top-down and bottom-up controls

Effects of fish predation propagate through aquatic food webs, where the classical grazing food chain and microbial loop are interwoven by trophic interactions. The overall impact on aquatic food webs is further complicated because fish may also exert bottom-up controls through nutrient regeneration. Yet, we still have limited information about cascading effects among fish, zooplankton, phytoplankton, and microbes. In this study, we performed a mesocosm experiment to evaluate effects of fish introduction on plankton communities. Six plots were set in factorial combination with fish introduction and rice straw plowing in a paddy field, and the experiment was continued for 4 weeks. Introduction of fish significantly increased chlorophyll a concentrations in smaller size fractions (<15 μm) and abundances of filamentous bacteria (>5 μm in length) and heterotrophic nanoflagellates in 3–15 μm fraction. Microbes in 0.8–3 μm fraction showed increasing but not significant trends in response to fish introduction. These results indicate cascading effects of fish predation operating via two pathways, one through grazing food chain and the other through microbial food web. Phytoplankton community compositions shifted in similar fashion in all plots until 1 week after fish introduction, and then diverged between plots with and without fish thereafter. Bottom-up effects of fish introduction were suggested by increases of total chlorophyll a and inedible phytoplankton species in response to fish introduction. This study provides an example of how fish predation regulates biomass and structure of phytoplankton and microbial communities.     

Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin

doi: 10.1111/j.1741‐2358.2010.00400.x
Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin Objective: This study evaluated the efficacy of disinfectants on the internal aspect of heat‐polymerised acrylic resin contaminated with microbial strains. Background: Dentures absorb oral fluids and become contaminated by different microorganisms. Methods: Two hundred and fifty rectangular specimens were made of heat‐polymerised acrylic resin, and then divided into five groups corresponding to the microbial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, S. mutans and Enterococcus faecalis). After contamination, the specimens were immersed in 1 and 2% sodium hypochlorite and 2% glutaraldehyde for periods of 5, 10 and 15 min. The specimens were placed into tubes containing different broths and incubated at 35°C and then visually analysed. Turbidity in the medium indicated microbial growth. The Fisher’s exact test was used in the analysis of the results. Results: The strain E. faecalis was the most resistant to the disinfectant solutions, and among them, glutaraldehyde was more effective than 2 and 1% hypochlorite for disinfection for 5 min; in the 10‐min period there were no differences between the disinfectants. In 15 min of immersion, 1% hypochlorite and glutaraldehyde were more effective than 2% hypochlorite. Conclusions: Disinfection for 10 min with 1% hypochlorite and glutaraldehyde is effective in disinfecting the internal aspect of heat‐polymerised acrylic resin.