Improved Microbial Production of Colominic Acid,a Homopolymer of N-Acetylneuraminic Acid

A culture medium has been devised for producing colominic acid in improved yields. Major improvements were obtained by using sorbitol as a source of carbon, by adding phosphate in high concentrations, and by supplementing a limited amount of yeast extract. E. coli O 16: Kl: HNM produced approximately 3000 µg/ml of colominic acid on cultivation at 37°C for 46 hr with a liquid medium consisting of sorbitol (2.0%), (NH₄)₂SO₄ (0.5%), K₂HPO₄ (1.4%), MgSO₄·7H₂O (0.05%), and yeast extract (0.05%).

Isolation and purification by deproteinization with ammonium sulfate, precipitation with ethanol, and by column chromatography on anion exchange resins resulted in a pure colominic acid preparation devoid of internal ester linkages.

In producing colominic acid, strains forming S-type colonies were more active than those forming R-type colonies.     

Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.     

Sustaining biological welfare for our future through consistent science

Physiological anthropology presently covers a very broad range of human knowledge and engineering technologies. This study reviews scientific inconsistencies within a variety of areas: sitting posture; negative air ions; oxygen inhalation; alpha brain waves induced by music and ultrasound; 1/f fluctuations; the evaluation of feelings using surface electroencephalography; Kansei; universal design; and anti-stress issues. We found that the inconsistencies within these areas indicate the importance of integrative thinking and the need to maintain the perspective on the biological benefit to humanity. Analytical science divides human physiological functions into discrete details, although individuals comprise a unified collection of whole-body functions. Such disparate considerations contribute to the misunderstanding of physiological functions and the misevaluation of positive and negative values for humankind. Research related to human health will, in future, depend on the concept of maintaining physiological functions based on consistent science and on sustaining human health to maintain biological welfare in future generations.     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.     

Excessive Cytokine Response to Rapid Proliferation of Highly Pathogenic Avian Influenza Viruses Leads to Fatal Systemic Capillary Leakage in Chickens

Highly pathogenic avian influenza viruses (HPAIVs) cause lethal infection in chickens. Severe cases of HPAIV infections have been also reported in mammals, including humans. In both mammals and birds, the relationship between host cytokine response to the infection with HPAIVs and lethal outcome has not been well understood. In the present study, the highly pathogenic avian influenza viruses A/turkey/Italy/4580/1999 (H7N1) (Ty/Italy) and A/chicken/Netherlands/2586/2003 (H7N7) (Ck/NL) and the low pathogenic avian influenza virus (LPAIV) A/chicken/Ibaraki/1/2005 (H5N2) (Ck/Ibaraki) were intranasally inoculated into chickens. Ty/Italy replicated more extensively than Ck/NL in systemic tissues of the chickens, especially in the brain, and induced excessive mRNA expression of inflammatory and antiviral cytokines (IFN-γ, IL-1β, IL-6, and IFN-α) in proportion to its proliferation. Using in situ hybridization, IL-6 mRNA was detected mainly in microglial nodules in the brain of the chickens infected with Ty/Italy. Capillary leakage assessed by Evans blue staining was observed in multiple organs, especially in the brains of the chickens infected with Ty/Italy, and was not observed in those infected with Ck/NL. In contrast, LPAIV caused only local infection in the chickens, with neither apparent cytokine expression nor capillary leakage in any tissue of the chickens. The present results indicate that an excessive cytokine response is induced by rapid and extensive proliferation of HPAIV and causes fatal multiple organ failure in chickens.     

Normal Lung Quantification in Usual Interstitial Pneumonia Pattern: The Impact of Threshold-based Volumetric CT Analysis for the Staging of Idiopathic Pulmonary Fibrosis

BackgroundAlthough several computer-aided computed tomography (CT) analysis methods have been reported to objectively assess the disease severity and progression of idiopathic pulmonary fibrosis (IPF), it is unclear which method is most practical. A universal severity classification system has not yet been adopted for IPF.ObjectiveThe purpose of this study was to test the correlation between quantitative-CT indices and lung physiology variables and to determine the ability of such indices to predict disease severity in IPF.MethodsA total of 27 IPF patients showing radiological UIP pattern on high-resolution (HR) CT were retrospectively enrolled. Staging of IPF was performed according to two classification systems: the Japanese and GAP (gender, age, and physiology) staging systems. CT images were assessed using a commercially available CT imaging analysis workstation, and the whole-lung mean CT value (MCT), the normally attenuated lung volume as defined from −950 HU to −701 Hounsfield unit (NL), the volume of the whole lung (WL), and the percentage of NL to WL (NL%), were calculated.ResultsCT indices (MCT, WL, and NL) closely correlated with lung physiology variables. Among them, NL strongly correlated with forced vital capacity (FVC) (r = 0.92, P <0.0001). NL% showed a large area under the receiver operating characteristic curve for detecting patients in the moderate or advanced stages of IPF. Multivariable logistic regression analyses showed that NL% is significantly more useful than the percentages of predicted FVC and predicted diffusing capacity of the lungs for carbon monoxide (Japanese stage II/III/IV [odds ratio, 0.73; 95% confidence intervals (CI), 0.48 to 0.92; P < 0.01]; III/IV [odds ratio. 0.80; 95% CI 0.59 to 0.96; P < 0.01]; GAP stage II/III [odds ratio, 0.79; 95% CI, 0.56 to 0.97; P < 0.05]).ConclusionThe measurement of NL% by threshold-based volumetric CT analysis may help improve IPF staging.     

A protein-protein interaction in magnetosomes: TPR protein MamA interacts with an Mms6 protein

Magnetosomes are membrane-enveloped bacterial organelles containing nano-sized magnetic particles, and function as a cellular magnetic sensor, which assist the cells to navigate and swim along the geomagnetic field. Localized with each magnetosome is a suite of proteins involved in the synthesis, maintenance and functionalization of the organelle, however the detailed molecular organization of the proteins in magnetosomes is unresolved. MamA is one of the most abundant magnetosome-associated proteins and is anchored to the magnetosome vesicles through protein-protein interactions, but the identity of the protein that interacts with MamA is undetermined. In this study, we found that MamA binds to a magnetosome membrane protein Mms6. Two different molecular masses of Mms6, 14.5-kDa and 6.0-kDa, were associated with the magnetosomes. Using affinity chromatography, we identified that the 14.5-kDa Mms6 interacts with MamA, and the interaction was further confirmed by pull-down, immunoprecipitation and size-exclusion chromatography assays. Prior to this, Mms6 was assumed to be strictly involved with biomineralizing magnetite; however, these results suggest that Mms6 has an additional responsibility, binding to MamA.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

Adenosine A2A receptor deficiency attenuates the somnogenic effect of prostaglandin D2

Sleep and Biological Rhythms -