Preparation of pH-sensitive poly(glycidol) derivatives with varying hydrophobicities: their ability to sensitize stable liposomes to pH

We have previously shown that modification with succinylated poly(glycidol) (SucPG) provides stable egg yolk phosphatidylcholine (EYPC) liposomes with pH-sensitive fusogenic property. Toward production of efficient pH-sensitive liposomes, in this study, we newly prepared three carboxylated poly(glycidol) derivatives with varying hydrophobicities by reacting poly(glycidol) with glutaric anhydride, 3-methylglutaric anhydride, and 1,2-cyclohexanedicarboxylic anhydride, respectively, designated as GluPG, MGluPG, and CHexPG. Correlation between side-chain structures of these polymers and their respective abilities to sensitize stable liposomes to pH was investigated. These polymers are soluble in water at neutral pH but became water-insoluble in weakly acidic conditions. The pH at which the polymer precipitated was higher in the order SucPG < GluPG < MGluPG < CHexPG, which is consistent with the number of carbon atoms of these polymers' side chains. Although CHexPG destabilized EYPC liposomes even at neutral pH, attachment of other polymers provided pH-sensitive properties to the liposomes. The liposomes bearing polymers with higher hydrophobicity exhibited more intense responses, such as content release and membrane fusion, at mildly acidic pH and achieved more efficient cytoplasmic delivery of membrane-impermeable dye molecules. As a result, modification with appropriate hydrophobicity, MGluPG, produced highly potent pH-sensitive liposomes, which might be useful for efficient cytoplasmic delivery of bioactive molecules, such as proteins and genes.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Conserved structural determinants in three-fingered protein domains

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.     

Protocol for the production of viable bimaternal mouse embryos

A reliable nuclear transfer method was first reported in 1983; it provided definite evidence that parthenogenetic embryos are lethal at early postimplantation in mammals. Subsequently, nuclear transfer has been extensively used as an important and versatile tool for investigating embryo and somatic-cell cloning and nucleo-cytoplasmic interactions. Further development of this technique has enabled the generation of bimaternal embryos containing two haploid sets of maternal genomes from female germ cells of different origins. By using a 2-d nuclear transfer system for oocyte reconstruction, viable mice can be produced solely from maternal genomes, without the participation of the paternal genome. This oocyte reconstruction system, as described in this protocol, could provide valuable guidelines for exploring the potential endowments of gametes and for conferring novel properties to them.     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.     

Optimal-foraging predator favors commensalistic Batesian mimicry

Background

Mimicry, in which one prey species (the Mimic) imitates the aposematic signals of another prey (the Model) to deceive their predators, has attracted the general interest of evolutionary biologists. Predator psychology, especially how the predator learns and forgets, has recently been recognized as an important factor in a predator–prey system. This idea is supported by both theoretical and experimental evidence, but is also the source of a good deal of controversy because of its novel prediction that in a Model/Mimic relationship even a moderately unpalatable Mimic increases the risk of the Model (quasi-Batesian mimicry).

Methodology/Principal Findings

We developed a psychology-based Monte Carlo model simulation of mimicry that incorporates a “Pavlovian” predator that practices an optimal foraging strategy, and examined how various ecological and psychological factors affect the relationships between a Model prey species and its Mimic. The behavior of the predator in our model is consistent with that reported by experimental studies, but our simulation''s predictions differed markedly from those of previous models of mimicry because a more abundant Mimic did not increase the predation risk of the Model when alternative prey were abundant. Moreover, a quasi-Batesian relationship emerges only when no or very few alternative prey items were available. Therefore, the availability of alternative prey rather than the precise method of predator learning critically determines the relationship between Model and Mimic. Moreover, the predation risk to the Model and Mimic is determined by the absolute density of the Model rather than by its density relative to that of the Mimic.

Conclusions/Significance

Although these predictions are counterintuitive, they can explain various kinds of data that have been offered in support of competitive theories. Our model results suggest that to understand mimicry in nature it is important to consider the likely presence of alternative prey and the possibility that predation pressure is not constant.     

Relationship between BMI and all-cause mortality in Japan: NIPPON DATA80

As body composition in Asian populations is largely different from Western populations, a healthy BMI could also differ between the two populations. Thus, further study is needed to determine whether a healthy BMI in Asians should be lower than Western populations, as recommended by the World Health Organization (WHO). We investigated the relationship between BMI and mortality in a sample of 8,924 Japanese men and women without stroke or heart disease. During 19 years of follow-up, 1,718 deaths were observed. We found a U-shaped relationship between BMI and fatal events. Risk of total mortality was highest in participants with BMI <18.5 kg/m(2) and lowest in participants with BMI 23.0-24.9 kg/m(2). These findings persisted even after excluding the first 5 years of follow-up with a focus on healthy participants who never smoked, were aged <70 years, and had total cholesterol (TC) levels >or=4.1 mmol/l (N=3712). For both the full sample and healthy participants, all-cause mortality risk did not differ between BMI ranges 21.0-22.9 and 23.0-24.9 kg/m(2). Our findings do not support the recent WHO implications that BMIs <23.0 kg/m(2) is healthy for Asians. Therefore, further studies are needed to identify an optimal BMI range for Asia.     

mKikGR,a Monomeric Photoswitchable Fluorescent Protein

The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.     

Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nociceptive neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Activation of TRPA1 by environmental irritants such as mustard oil, allicin and acrolein causes acute pain. However, the endogenous ligands that directly activate TRPA1 remain elusive in inflammation. Here, we show that a variety of inflammatory mediators (15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), nitric oxide (NO), hydrogen peroxide (H(2)O(2)), and proton (H(+))) activate human TRPA1 heterologously expressed in HEK cells. These inflammatory mediators induced robust Ca(2+) influx in a subset of mouse DRG neurons. The TRP channel blocker ruthenium red almost completely inhibited neuronal responses by 15d-PGJ(2) and NO, but partially suppressed responses to H(2)O(2) and H(+). Functional characterization of site-directed cysteine mutants of TRPA1 in combination with labeling experiments using biotinylated 15d-PGJ(2) demonstrated that modifications of cytoplasmic N-terminal cysteines (Cys421 and Cys621) were responsible for the activation of TRPA1 by 15d-PGJ(2). In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H(2)O(2), the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ(2). Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H(+) as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.     

Mycolyltransferase-mediated glycolipid exchange in Mycobacteria

Trehalose dimycolate (TDM), also known as cord factor, is a major surface glycolipid of the cell wall of mycobacteria. Because of its potent biological functions in models of infection, adjuvancy, and immunotherapy, it is important to determine how its biosynthesis is regulated. Here we show that glucose, a host-derived product that is not readily available in the environment, causes Mycobacterium avium to down-regulate TDM expression while up-regulating production of another major glycolipid with immunological roles in T cell activation, glucose monomycolate (GMM). In vitro, the mechanism of reciprocal regulation of TDM and GMM involves competitive substrate selection by antigen 85A. The switch from TDM to GMM biosynthesis occurs near the physiological concentration of glucose present in mammalian hosts. We further demonstrate that GMM is produced in vivo by mycobacteria growing in mouse lung. These results establish an enzymatic pathway for GMM production. More generally, these observations provide a specific enzymatic mechanism for dynamic alterations of cell wall glycolipid remodeling in response to the transition from noncellular to cellular growth environments, including factors that are monitored by the host immune system.