Structural characterization of MG and pre-MG states of proteins by MD simulations,NMR, and other techniques

Almost all proteins fold via a number of partially structured intermediates such as molten globule (MG) and pre-molten globule states. Understanding the structure of these intermediates at atomic level is often a challenge, as these states are observed under extreme conditions of pH, temperature, and chemical denaturants. Furthermore, several other processes such as chemical modification, site-directed mutagenesis (or point mutation), and cleavage of covalent bond of natural proteins often lead to MG like partially unfolded conformation. However, the dynamic nature of proteins in these states makes them unsuitable for most structure determination at atomic level. Intermediate states studied so far have been characterized mostly by circular dichroism, fluorescence, viscosity, dynamic light scattering measurements, dye binding, infrared techniques, molecular dynamics simulations, etc. There is a limited amount of structural data available on these intermediate states by nuclear magnetic resonance (NMR) and hence there is a need to characterize these states at the molecular level. In this review, we present characterization of equilibrium intermediates by biophysical techniques with special reference to NMR.     

Comparative immunomodulatory properties of adipose‐derived mesenchymal stem cells conditioned media from BALB/c,C57BL/6, and DBA mouse strains

Adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) have been shown to be capable of differentiating into multiple cell type and exert immunomodulatory effects. Since the selection of ideal stem cell is apparently crucial for the outcome of experimental stem cell therapies, therefore, in this study we compared AD‐MSCs conditioned media (CM) from BALB/c, C57BL/6, and DBA mouse strains. No significant difference was found in the morphology, cell surface markers, in vitro differentiation and proliferation potentials of AD‐MSCs isolated from C57BL/6, BALB/c, and DBA mice. The immunological assays showed some variation among the strains in the cytokines, nitric oxide (NO), and indoleamine 2,3‐dioxygenase (IDO) production and immunomodulatory effects on splenocytes functions. Our results indicated a suppression of splenocytes proliferation in the presence of AD‐MSC CM from the three inbred mouse strains. However, BALB/c CM exerted a higher suppression of splenocytes proliferation. AD‐MSCs isolated from C57BL/6 and BALB/c mice produced higher levels of TGF‐β than those from DBA mice. Furthermore, IL‐17 and IDO production was higher in AD‐MSCs isolated from BALB/c mice. Our results indicated an increased production of TGF‐β, IL‐4, IL‐10, NO, and IDO by splenocytes in response to CM from BALB/c AD‐MSCs. In conclusion, our results showed that the immunomodulatory properties of mouse AD‐MSCs is strain‐dependent and this variation should be considered during selection of appropriate stem cell source for in vivo experiments and stem cell therapy strategies. J. Cell. Biochem. 114: 955–965, 2013. © 2012 Wiley Periodicals, Inc.     

Gastrokine 1 regulates NF‐κB signaling pathway and cytokine expression in gastric cancers

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF‐κB, COX‐2, and cytokines in GKN1‐transfected AGS cells and shGKN1‐transfected HFE‐145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1‐transfected AGS cells, we observed inactivation and reduced expression of NF‐κB and COX‐2, whereas shGKN1‐transfected HFE‐145 cells showed activation and increased expression of NF‐κB and COX‐2. GKN1 expression induced production of inflammatory cytokines including IL‐8 and ‐17A, but decreased expression of IL‐6 and ‐10. We also found IL‐17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE‐145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF‐κB signaling pathway and cytokine expression. J. Cell. Biochem. 114: 1800–1809, 2013. © 2013 Wiley Periodicals, Inc.     

Enhanced tolerance to drought and salt stresses in transgenic faba bean (Vicia faba L.) plants by heterologous expression of the PR10a gene from potato

Key message

We report for the first time that expression of potato PR10a gene in faba bean causes enhanced tolerance to drought and salinity.

Abstract

Grain legumes such as soybean (Glycine max L. Merrill), pea (Pisum sativum L.) and faba bean (Vicia faba L.) are staple sources of protein for human and animal nutrition. Among grain legumes, faba bean is particularly sensitive to abiotic stress (in particular osmotic stress due to lack of water or enhanced soil salinity) and often suffers from severe yield losses. Many stress responsive genes have been reported with an effect on improving stress tolerance in model plants. Pathogenesis-related proteins are expressed by all plants in response to pathogen infection and, in many cases, in response to abiotic stresses as well. The PR10a gene isolated from the potato cultivar Desiree was selected for this study due to its role in enhancing salt and/or drought tolerance in potato, and transferred into faba bean cultivar Tattoo by Agrobacterium tumefaciens-mediated transformation system based upon direct shoot regeneration after transformation of meristematic cells derived from embryo axes. The transgene was under the control of the constitutive mannopine synthase promoter (p-MAS) in a dicistronic binary vector, which also contained luciferase (Luc) gene as scorable marker linked by internal ribosome entry site elements. Fertile transgenic faba bean plants were recovered. Inheritance and expression of the foreign genes were demonstrated by PCR, RT-PCR, Southern blot and monitoring of Luciferase activity. Under drought condition, after withholding water for 3 weeks, the leaves of transgenic plants were still green, while non-transgenic plants (WT) wilted and turned brown. Twenty-four hours after re-watering, the leaves of transgenic plants remained green, while WT plants did not recover. Moreover, the transgenic lines displayed higher tolerance to NaCl stress. Our results suggested that introducing a novel PR10a gene into faba bean could be a promising approach to improve its drought and salt tolerance ability, and that MAS promoter is not only constitutive, but also wound-, auxin/cytokinin- as well as stress-inducible.     

Chiral Separation of the Clinically Important Compounds Fucose and Pipecolic Acid Using CE: Determination of the Most Effective Chiral Selector

In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC‐Cl) and 5‐amino‐2‐naphthalene‐sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10‐mM β‐cyclodextrin was able to separate the enantiomers of ANSA‐DL‐fucose and the polymeric surfactant poly(sodium N‐undecanoyl‐LL‐leucine‐valinate) was able to separate the enantiomers of FMOC‐DL‐pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D‐alanine tert‐butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. Chirality 25:556–560, 2013. © 2013 Wiley Periodicals, Inc.     

Fecal Influenza in Mammals: Selection of Novel Variants

In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2^E627K, HA^F128V, HA^F454L, or HA^H300P variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.     

Bacillus cereus adhesion: Real time investigation of the effect on the chemistry of industrial stainless steel

The initial microorganism adhesion on substrate is an important step for the biofilm formation. The surface properties of the stainless steel and B. cereus were characterized by the sessile drop technique. Moreover, the physicochemical properties of surface adhesion and the impact of bio adhesion to the stainless steel were determined at different time of contact (2, 4, 7, 9 and 24 h). The results showed that the strain was hydrophilic (Giwi = 3.37 mJ/m²), whereas the substratum has hydrophobic character (Giwi = ?57.6 mJ/m²). Stainless steel surface presents a weak electron-donor character (γ^? = 4.1 mJ/m²) conversely to B. cereus that presents an important parameter (γ^? = 31.6 mJ/m²). The bio adhesion was investigated at different time of contact. The data analysis after 2 h, confirmed the adhesion of B. cereus with an amount of 10 cfu/cm² which increased to 1.210⁴ cfu/cm² after 24 h. Interestingly, despite the difference of hydropohbicity, the interaction between B. cereus and substratum was favored by the thermodynamic aspect of adhesion (ΔG_adhesion < 0). Interestingly, the study of the effect of B. cereus adhesion on the stainless steel has revealed that, the substratum becomes hydrophilic (θ° = 41.3, ΔGiwi = 39.6 mJ/m²) and highly electron donor (Γ^? = 52.9 mJ/m²) after 2 h of bio adhesion.     

Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal

Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5–6.5, and CBHII: 46 °C, pH 4.8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion in the parallel batch reaction was only ~5 % by weight. Also, a synergistic effect, achieving ~27 % substrate conversion, was obtained by addition of endo-1,4-β-d-glucanase. The synergistic effect was only obtained with product removal. By using pure, monoactive enzymes, the work illustrates the profound gains achievable by intermittent product removal during cellulose hydrolysis.     

The population of Odonata (dragonflies) in small tropical rivers with reference to asynchronous growth patterns

The odonate larval communities in three small rivers in Penang Island were studied. More species of dragonflies were found in the Botanical Garden and Titi Teras rivers (13 and 11 respectively) of relatively similar environmental parameters. Fewer (nine) dragonfly species were collected from the Youth Park River which has a lower dissolved oxygen (DO) and a higher biological oxygen demand (BOD), conductivity and turbidity. A mixture of sand, gravel and pebble substrate of Botanical Garden River with dense growth of submerged Hydrilla, grasses and Cladias (Araceae) provided suitable habitats for the dragonflies. The sandy substrate and relatively fast flowing water of Titi Teras River was highly preferred by gomphids. In the Youth Park River, the small community of dragonfly larvae was dominated by tolerant Pseudagrion rubriceps, P. microcephalum, Orthetrum chrysis and Crocothemis servilia. Based on the larval instar distribution of Ictinogomphus decoratus and O. chrysis, very asynchronous populations of these dragonflies occurred in each river. Young larvae were continuously introduced into the populations resulting in undulating growth rate curves. The growth rates of these two species were higher in the Titi Teras River when compared to those in other rivers. Density-dependent mortality, asynchronous cannibalism and fish predation could play important roles in regulating the larval dragonfly population in these rivers.     

A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.