Effect of Polyethylene Glycol on the Thermal Stability of Green Fluorescent Protein

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70-95 degrees C. Thermal stability was expressed in decimal reduction time. It was observed that the D-values obtained were almost constant for temperatures of 85, 90, and 95 degrees C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75 degrees C, PEG 600 and 4,000 g/mol stabilized GFP. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 252-256, 2010

Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach

Clavulanic acid (CA) is a beta-lactam antibiotic that alone exhibits only weak antibacterial activity, but is a potent inhibitor of beta-lactamases enzymes. For this reason it is used as a therapeutic in conjunction with penicillins and cephalosporins. However, it is a well-known fact that it is unstable not only during its production phase, but also during downstream processing. Therefore, the main objective of this study was the evaluation of CA long-term stability under different conditions of pH and temperature, in the presence of variable levels of different salts, so as to suggest the best conditions to perform its simultaneous production and recovery by two-phase polymer/salt liquid-liquid extractive fermentation. To this purpose, the CA stability was investigated at different values of pH (4.0-8.0) and temperature (20-45 degrees C), and the best conditions were met at a pH 6.0-7.2 and 20 degrees C. Its stability was also investigated at 30 degrees C in the presence of NaCl, Na(2)SO(4), CaCl(2) and MgSO(4) at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 6.5). All salts led to increased CA instability with respect to the buffer alone, and this effect decreased in following sequence: Na(2)SO(4) > MgSO(4) > CaCl(2) > NaCl. Kinetic and thermodynamic parameters of CA degradation were calculated adopting a new model that took into consideration the equilibrium between the active and a reversibly inactivated form of CA after long-time degradation. (C) 2009 Elsevier B.V. All rights reserved.

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence or the ratio of renewed volume to total volume (R), the Urea feeding time (t(f)) and the number of successive repealed fed-batch cycles on the maximum cell concentration (X(m)), cell productivity (P(x)), nitrogen-to-cell conversion yield (Y(x/n)), maximum specific growth rate (mu(m)) and protein content of, dry biomass. The experimental results demonstrated chat R=0.80 and t(f) = 6d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X(m) = 2101 +/- 113 mg L(-1), P(x) = 219 +/- 13 mg L(-1) d(-1) and Y(x/n) = 10.3 +/- 0.8,g g(-1)). (C) 2008 Elsevier B.V. All rights reserved.

Protein partitioning in poly(ethylene glycol)/sodium polyacrylate aqueous two-phase systems

The partition of hemoglobin, lysozyme and glucose-6-phospate dehydrogenase (G6PDH) in a novel inexpensive aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The effect of NaCl and Na2SO4, pH and PEG molecular size on the partitioning has been studied. At high pH (above 9), hemoglobin partitions strongly to the PEG-phase. Although some precipitation of hemoglobin occurs, high recovery values are obtained particularly for lysozyme and G6PDH. The partitioning forces are dominated by the hydrophobic and electrochemical (salt) effects, since the positively charged lysozyme and negatively charged G6PDH partitions to the non-charged PEG and the strongly negatively charged polyacrylate enriched phase, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Investigation of charged polymer influence on green fluorescent protein thermal stability

Methods of stabilization and formulation of proteins are important in both biopharmaceutical and biocatalysis industries. Polymers are often used as modifiers of characteristics of biological macromolecules to improve the biochemical activity and stability of proteins or drug bioavailability. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Relative thermal stability was undertaken by incubation of GFP at varying temperatures and GFP fluorescence was used as a reporter for unfolding. At 80 degrees C, DEAE-dextran did not have any effect on GFP fluorescence, indicating that it does not confer stability.

Distinct conformational properties determined by implicit and explicit representation of protein-solvent interactions. An analytical and computer simulation study

In the protein folding problem, solvent-mediated forces are commonly represented by intra-chain pairwise contact energy. Although this approximation has proven to be useful in several circumstances, it is limited in some other aspects of the problem. Here we show that it is possible to achieve two models to represent the chain-solvent system. one of them with implicit and other with explicit solvent, such that both reproduce the same thermodynamic results. Firstly, lattice models treated by analytical methods, were used to show that the implicit and explicitly representation of solvent effects can be energetically equivalent only if local solvent properties are time and spatially invariant. Following, applying the same reasoning Used for the lattice models, two inter-consistent Monte Carlo off-lattice models for implicit and explicit solvent are constructed, being that now in the latter the solvent properties are allowed to fluctuate. Then, it is shown that the chain configurational evolution as well as the globule equilibrium conformation are significantly distinct for implicit and explicit solvent systems. Actually, strongly contrasting with the implicit solvent version, the explicit solvent model predicts: (i) a malleable globule, in agreement with the estimated large protein-volume fluctuations; (ii) thermal conformational stability, resembling the conformational hear resistance of globular proteins, in which radii of gyration are practically insensitive to thermal effects over a relatively wide range of temperatures; and (iii) smaller radii of gyration at higher temperatures, indicating that the chain conformational entropy in the unfolded state is significantly smaller than that estimated from random coil configurations. Finally, we comment on the meaning of these results with respect to the understanding of the folding process. (C) 2009 Elsevier B.V. All rights reserved.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the Delta ddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The Delta ddbA mutation can genetically interact with uvsB(ATR), atmA(ATM), nkuA(KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne`s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP:DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

Enhanced skin penetration of P20 phosphopeptide using protein transduction domains

Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1 mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4 h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent. (c) 2007 Elsevier B.V. All rights reserved.

Role of phospholipase C and protein kinase C in Aspergillus nidulans during growth on pectin or glucose: Effects on germination and duplication cycle

The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.

Circulating Interleukin-6 and High-Sensitivity C-Reactive Protein Decrease After Periodontal Therapy in Otherwise Healthy Subjects

Background: Periodontal disease has been associated with many chronic inflammatory systemic diseases, and a common chronic inflammation pathway has been suggested for these conditions. However, few studies have evaluated whether periodontal disease, in the absence of other known inflammatory conditions and smoking, affects circulating markers of chronic inflammation. This study compared chronic inflammation markers in control individuals and patients with periodontal disease and observed whether non-surgical periodontal therapy affected inflammatory disease markers after 3 months. Methods: Plasma and serum of 20 controls and 25 patients with periodontal disease were obtained prior to and 3 months after non-surgical periodontal therapy. All patients were non-smokers, they did not use any medication, and they had no history or detectable signs and symptoms of systemic diseases. Periodontal and systemic parameters included probing depth, bleeding on probing, clinical attachment level, hematologic parameters, as well as the following inflammatory markers: interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), CD40 ligand, monocyte chemoattractant protein (MCP)-1, soluble P-selectin (sP-selectin), soluble vascular adhesion molecule (sVCAM)-1, and soluble intercellular adhesion molecule (sICAM)-1. Results: There were no differences in the hematologic parameters of the patients in the control and periodontal disease groups. Among the tested inflammatory markers, IL-6 concentrations were higher in the periodontal disease group at baseline compared to the controls (P=0.006). Therapy was highly effective (P<0.001 for all the analyzed clinical parameters), and a decrease in circulating IL-6 and hs-CRP concentrations was observed 3 months after therapy (P=0.001 and P=0.006, respectively). Our results also suggest that the CD40 ligand marker may have been different in the control and periodontal disease groups prior to the therapy (P=0.009). Conclusions: In apparently otherwise healthy patients, periodontal disease is associated with increased circulating concentrations of IL-6 and hs-CRP, which decreased 3 months after non-surgical periodontal therapy. With regard to the CD40 ligand, MCP-1, sP-selectin, sVCAM-1, and sICAM-1, no changes were seen in the periodontal disease group between baseline and 3 months after therapy. J Periodontol 2009;80:594-602.

A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1)

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue dagger.

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, b- cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5 degrees C and 40% at 25 degrees C after 8 months. The lipase dried with 10% of beta-cyclodextrin retained 72% of activity at 5 degrees C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.

Influence of Plasma Protein Binding on Pharmacodynamics: Estimation of In Vivo Receptor Affinities of beta Blockers Using a New Mechanism-Based PK-PD Modelling Approach

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B),(vivo)). These values were compared with in vitro affinities (K(B),(vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B),(vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B),(vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B),(vivo)-K(B),(vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B),(vivo)/K(B),(vitro) similar to 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3816-3828, 2009

Deconstructing Tick Saliva NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-alpha while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of similar to 110pmol/mu l) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) similar to 100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

Research Quality, Publications and Impact in Hydraulic Engineering into the 21st Century. Publish or Perish, Commercial versus Open Access, Internet versus Libraries

One of the main objectives of the first International Junior Researcher and Engineer Workshop on Hydraulic Structures is to provide an opportunity for young researchers and engineers to present their research. But a research project is only completed when it has been published and shared with the community. Referees and peer experts play an important role to control the research quality. While some new electronic tools provide further means to disseminate some research information, the quality and impact of the works remain linked with some thorough expert-review process and the publications in international scientific journals and books. Importantly unethical publishing standards are not acceptable and cheating is despicable.