Isoprostane nomenclature: Inherent problems may cause setbacks for the development of the isoprostanoid field

Do we have to bother about the isoprostane nomenclature? The widely accepted IUPAC isoprostane nomenclature provides an unambiguous and systematic terminology to name all theoretical possible isoprostanes. However, the currently accepted nomenclature system provides an unnatural framework which is not well suited to address certain biologically relevant questions. Artificial categorization of isoprostanoids into prostanoid families disrupts prostaglandin-ring core structures needed to describe biogenetic precursor–product relationships. In addition, the IUPAC system defines isoprostanoid families which comprise chemically heterogeneous isoprostanoids which largely differ in their physicochemical properties from those of the corresponding prostaglandins. As a result of this, alternative nomenclature systems such as the phytoprostane nomenclature system overcoming some inherent problems of the IUPAC nomenclature are still in use. However, different naming of isoprostanoids especially the classification of prostanoid family names has created considerable confusion. Therefore, a cautionary note on the current use of different nomenclature systems is necessary.     

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.     

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.     

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

Background

The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).

Results

We show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.

Conclusion

Our data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.     

EGFR/Met association regulates EGFR TKI resistance in breast cancer

Breast cancers show a lack of response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), despite 30% of tumors expressing EGFR. The mechanism of this resistance is unknown; however, we have recently shown that Met kinase activity compensates for loss of EGFR kinase activity in cell culture models. Met has been implicated in the pathogenesis of breast tumors and therefore may cooperate with EGFR for tumor growth. Here we have found that EGFR phosphorylation and cell proliferation is in part regulated by Met expression. In addition, we found that Met constitutive phosphorylation occurred independent of the Met ligand hepatocyte growth factor (HGF). Ligand-independent Met phosphorylation is mediated by Met amplification, mutation, or overexpression and by Met interaction with other cell surface molecules. In SUM229 breast cancer cells, we found that Met was not amplified or mutated, however it was overexpressed. Met overexpression did not directly correlate with ligand-independent Met phosphorylation as the SUM229 cell line was the only Met expressing breast cancer line with constitutive Met phosphorylation. Interestingly, Met expression did correlate with EGFR expression and we identified an EGFR/Met complex via co-immunoprecipitation. However, we only observed Met constitutive phosphorylation when c-Src also was part of this complex. Ligand-independent phosphorylation of Met was decreased by down regulating EGFR expression or by inhibiting c-Src kinase activity. Lastly, inhibiting EGFR and Met kinase activities resulted in a synergistic decrease in cell proliferation, supporting the idea that EGFR and Met functionally, as well as physically interact in breast cancer cells to regulate response to EGFR inhibitors.     

Effect of light exposure on in vitro germination and germ tube growth of eight species of rust fungi

The effects of light on urediniospore germination and germ tube elongation was studied with eight species of rust fungi that infect ornamental plants or row crops. Exposure of six species of fungi to cool white fluorescent light at 400 or 600 micromol s(-1) m(-2) for 24 h significantly reduced germination with largest decreases typically observed at 600 micromol s(-1) m(-2). Germination and germ tube elongation did not recover during 24 h dark incubation after 18 h exposure to fluorescent light at 600 micromol s(-1) m(-2), indicating the effects were not reversible. Germ tube elongation of all fungi was negatively affected by increased length of exposure to fluorescent light. Increased exposure to fluorescent light differentially affected germination of the fungi with Puccinia hemerocallidis, Phakopsora pachyrhizi, Pucciniastrum vaccinii and Puccinia menthae negatively affected and Puccinia sorghi, Puccinia triticina, Puccinia pelargonii-zonalis and Puccinia iridis relatively unaffected in 10 h incubation. Exposure of Ph. pachyrhizi and P. triticina urediniospores to sunlight rapidly reduced germination and germ tube elongation with no germination observed for Ph. pachyrhizi after 2.5 h. Germ tube elongation but not germination of hydrated urediniospores of Ph. pachyrhizi and P. triticina was significantly reduced compared to dry urediniospores exposed to 10 h fluorescent light followed by 24 h dark incubation. Exposure to fluorescent light (all fungi) or sunlight (two fungi) negatively affected urediniospore germ tube elongation. Differences observed in urediniospore germination between fungi suggest some species have co-evolved with their host for differing light conditions. Our data suggests exposure of urediniospores to strong light could inactivate rust fungi on plant surfaces or in the atmosphere.     

Differential Impact of Lipoxygenase 2 and Jasmonates on Natural and Stress-Induced Senescence in Arabidopsis

Jasmonic acid and related oxylipins are controversially discussed to be involved in regulating the initiation and progression of leaf senescence. To this end, we analyzed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis (Arabidopsis thaliana). Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes, with the strongest increase of jasmonic acid after dark treatment. Arabidopside content only increased considerably in response to sorbitol treatment. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNA interference (RNAi) plants were generated, which constitutively produce jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered, suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase 2-RNAi lines and the allene oxid synthase-deficient mutant dde2 were less sensitive to sorbitol than the wild type, indicating that oxylipins contribute to the response to sorbitol stress.Senescence is an important, highly regulated process at the end of development. Senescence is characterized by breakdown of organelles and molecules, export and transport of these nutrients to other organs/parts of the organism, and finally programmed cell death of the senescing organ.The process of senescence has been intensively studied in leaves, and morphological as well as molecular changes in senescing leaves have been described. Yellowing as a consequence of chlorophyll and chloroplast degradation is the most obvious process during natural leaf senescence. In addition, gene expression changes dramatically during senescence. Some senescence-associated genes (SAG, SEN) have been reported that are induced during this process, and several of the encoded proteins function in macromolecule degradation, detoxification and defense metabolism, or signal transduction (Gepstein et al., 2003). Based on the degradation of chloroplasts and macromolecules, leaf metabolism changes from carbon assimilation to catabolism (Lim et al., 2007).The initiation and progression of senescence is regulated by endogenous as well as exogenous factors. Among the endogenous factors, the developmental status of the organ and of the whole plant (e.g. age and progress in flowering and seed production) has a great impact on the process of senescence. Different stress factors such as pathogen attack, drought, osmotic stress, heat, cold, ozone, UV light, and shading can induce or accelerate senescence (Quirino et al., 2000). Phytohormones are very important regulators that integrate information about the developmental status and the environmental factors. Cytokinins are antagonistic signals and delay senescence. Endogenous levels of cytokinins decrease during senescence, and exogenous application and transgenic approaches, enhancing endogenous levels of these compounds, lead to delayed senescence (Gan and Amasino, 1995). In contrast, the gaseous phytohormone ethylene is known to induce and accelerate senescence (John et al., 1995). There are also several indications that abscisic acid modulates senescence (van der Graaff et al., 2006). The roles of other phytohormones/signaling compounds such as auxin, salicylic acid, and jasmonates are less clear (Lim et al., 2007).Jasmonates are oxylipin signaling molecules derived from linolenic acid. The term jasmonates comprises 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives such as the methyl ester and amino acid conjugates of JA. One of the first biological activities described for these compounds was the promotion of senescence in oat (Avena sativa) leaves by methyl jasmonate (MeJa) isolated from Artemisia absinthium (Ueda and Kato, 1980). Later on, the induction of senescence-like phenotypes by exogenous application of MeJa was also found in other plant species (Ueda and Kato, 1980; Weidhase et al., 1987a; He et al., 2002). On the molecular level, this senescence-promoting effect of MeJa is accompanied by chlorophyll loss and decreases in Rubisco and photosynthesis (Weidhase et al., 1987a, 1987b). In addition, expression of some senescence-up-regulated genes is also responsive to JA; examples are SEN1, SEN4, SEN5, SAG12, SAG14, and SAG15 (Park et al., 1998; Schenk et al., 2000; He et al., 2002). Due to the results described above, jasmonates have been described for decades as compounds with senescence-promoting activities, while the function of these compounds in natural senescence in planta was critically discussed (Parthier, 1990; Sembdner and Parthier, 1993; Creelman and Mullet, 1997; Wasternack, 2007; Balbi and Devoto, 2008; Reinbothe et al., 2009). Additional indications for a role of jasmonates in regulating senescence are the transient up-regulation of expression of some enzymes involved in JA biosynthesis, such as allene oxide synthase (AOS) and OPDA reductase 3 (OPR3), and the increase in JA levels during natural senescence (He et al., 2002; van der Graaff et al., 2006). Furthermore, alterations in natural and induced senescence have been reported for some mutants with defects in the JA pathway. The mutant coi1, which is impaired in JA signaling, exhibited delayed chlorophyll loss upon dark incubation of detached leaves (Castillo and Leon, 2008). Plants with reduced expression of the 3-ketoacyl-CoA-thiolase KAT2, which is involved in β-oxidation and JA production, showed delayed yellowing during natural senescence and upon dark incubation of detached leaves (Castillo and Leon, 2008).However, there are also several reports that cast doubt on an important function of JA in senescence. For most mutants in JA biosynthesis or signaling, no differences in natural senescence are apparent (He et al., 2002; Schommer et al., 2008). In addition, mutants defective in the expression of AOS or OPR3 do not show altered senescence-like phenotypes upon dark treatment (Schommer et al., 2008; Kunz et al., 2009). It has to be taken into consideration that the knockout in these mutants has pleiotrophic effects during whole plant development. For example, the leaves of plants with reduced expression of the lipase DGL or of OPR3 are larger (Hyun et al., 2008). In addition, several knockout mutants defective in JA biosynthesis or signaling do not produce fertile flowers (Feys et al., 1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001; von Malek et al., 2002). These changes in development might affect other developmental processes such as senescence.To investigate the function of jasmonates in senescence in more detail, we compared the oxylipin profile of wild-type leaves during natural senescence and upon stress induction of senescence-like phenotypes. The analysis of lipoxygenase 2 (LOX2)-RNA interference (RNAi) plants, which produce low basal levels of oxylipins but are impaired in the accumulation of OPDA and JA during senescence or in response to stress, indicates that 13-LOX products are not necessary for natural senescence or dark-induced chlorophyll loss but are involved in the response to sorbitol.     

Reducing the Risk of Contamination of Sterile Parenteral Products via Ready-to-Use Closure Components

The preparation of sterile parenteral products requires careful control of all ingredients, materials, and processes to ensure the final product has the identity and strength, and meets the quality and purity characteristics that it purports to possess. Contamination affecting these critical properties of parenteral products can occur in many ways and from many sources. The use of closures supplied by manufacturers in a ready-to-use state can be an effective method for reducing the risk of contamination and improving the quality of the drug product. This article will address contamination attributable to elastomeric container closure components and the regulatory requirements associated with container closure systems. Possible contaminants, including microorganisms, endotoxins, and chemicals, along with the methods by which these contaminants can enter the product will be reviewed. Such methods include inappropriate material selection, improper closure preparation processes, compromised container closure integrity, degradation of closures, and leaching of compounds from the closures.