The Membrane‐Associated Protein,Supervillin, Accelerates F‐Actin‐Dependent Rapid Integrin Recycling and Cell Motility

In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F‐actin‐ and myosin II‐binding protein that tightly associates with signaling proteins in cholesterol‐rich, ‘lipid raft’ membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin‐knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed‐end inhibitor, cytochalasin D, suggests that both treatments affect actin‐dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal‐regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F‐actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin‐based cell motility.     

Developing a management procedure robust to uncertainty for southern bluefin tuna: a somewhat frustrating struggle to bridge the gap between ideals and reality

Fisheries management is conducted to achieve sustainable use of fishery resources, mainly through regulation of fishing activities. For almost a decade, the Commission for the Conservation of Southern Bluefin Tuna (CCSBT) struggled to reach agreement on a total allowable catch (TAC) for southern bluefin tuna (SBT) because of stock assessment uncertainties. To address this, in 2002 the CCSBT commenced development of a management procedure (MP), a pre-agreed set of rules to determine how the TAC will be adjusted as new monitoring data become available. The CCSBT Scientific Committee tested various candidate MPs using operating models which simulate fish population and fishery dynamics as well as incorporate process, observation, and model uncertainties. Candidate MPs were evaluated using performance measures related to the following management objectives: maximize catches, avoid stock collapse, and minimize interannual catch variation. Of the MPs explored, some relied solely on empirical data [i.e., adjusted TAC based on catch per unit effort (CPUE) trends], whereas others were more complicated, based on population models. In 2005, the CCSBT adopted a model-based MP that realized a moderate catch with low variability and avoided stock collapse. This MP struck a compromise between the risk-prone and risk-averse standpoints of the different stakeholders. However, despite this concerted scientific effort, the MP was not implemented because, shortly after its adoption, it became evident that historical catches may have been substantially underreported. This complication necessitates returning to near the beginning of the development process. MP approaches have various advantages and challenges to be explored further. However, it is essential to lessen human-introduced uncertainty (such as catch misreporting) by enhanced enforcement, and to increase management robustness to biological uncertainties by implementing MPs.     

Cloning and targeted disruption of two lipopolysaccharide biosynthesis genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by site-directed mutagenesis

The emergence of antibiotic resistance in bacterial pathogens poses a great challenge to public health and emphasizes the need for new antimicrobial targets. The recent development of microbial genomics and the availability of genome sequences allows for the identification of essential genes which could be novel and potential targets for antibacterial drugs. However, these predicted targets need experimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the pathogen Pseudomonas aeruginosa PAO1 using insertion duplication. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP-glucose (heptosyl) LPS α-1,3-glucosyltransferase were selected as putative target candidates for the gene disruption experiments using plasmid insertion mutagenesis to determine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome resulted in lack of recovery of antibiotic-resistant colonies suggesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

Very strong UV-A light temporally separates the photoinhibition of photosystem II into light-induced inactivation and repair

When organisms that perform oxygenic photosynthesis are exposed to strong visible or UV light, inactivation of photosystem II (PSII) occurs. However, such organisms are able rapidly to repair the photoinactivated PSII. The phenomenon of photoinactivation and repair is known as photoinhibition. Under normal laboratory conditions, the rate of repair is similar to or faster than the rate of photoinactivation, preventing the detailed analysis of photoinactivation and repair as separate processes. We report here that, using strong UV-A light from a laser, we were able to analyze separately the photoinactivation and repair of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Very strong UV-A light at 364 nm and a photon flux density of 2600 micromol photons m(-2) s(-1) inactivated the oxygen-evolving machinery and the photochemical reaction center of PSII within 1 or 2 min before the first step in the repair process, namely, the degradation of the D1 protein, occurred. During subsequent incubation of cells in weak visible light, the activity of PSII recovered fully within 30 min and this process depended on protein synthesis. During subsequent incubation of cells in darkness for 60 min, the D1 protein of the photoinactivated PSII was degraded. Further incubation in weak visible light resulted in the rapid restoration of the activity of PSII. These observations suggest that very strong UV-A light is a useful tool for the analysis of the repair of PSII after photoinactivation.     

Changes of cerebral blood oxygenation and optical pathlength during activation and deactivation in the prefrontal cortex measured by time-resolved near infrared spectroscopy

To determine the alterations in optical characteristics and cerebral blood oxygenation (CBO) during activation and deactivation, we evaluated the changes in mean optical pathlength (MOP) and CBO induced by a verbal fluency task (VFT) and driving simulation in the right and left prefrontal cortex (PFC), employing a newly developed time-resolved near infrared spectroscopy, which allows quantitative measurements of the evoked-CBO changes by determining the MOP with a sampling time of 1 s. The results demonstrated differences in MOP in the foreheads with the subjects and wavelength; however, there was no significant difference between the right and left foreheads (p > 0.05). Also, both the VFT and driving simulation task did not affect the MOP significantly as compared to that before the tasks (p > 0.05). In the bilateral PFCs, the VFT caused increases of oxyhemoglobin and total hemoglobin associated with a decrease of deoxyhemoglobin, while the driving simulation task caused decreases of oxyhemoglobin and total hemoglobin associated with an increase of deoxyhemoglobin; there were no significant differences in evoked-CBO changes between the right and left PFC. The present results will be useful for quantitative measurement of hemodynamic changes during activation and deactivation in the adults by near infrared spectroscopy.     

Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress

Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance.     

Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution

A biased mutation-assembling method—that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Periostin is an extracellular matrix protein required for eruption of incisors in mice

A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin-/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin-/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone.