Insoluble Cell Research Articles

Antioxidant Activity of Free and Bound Compounds in Quinoa (Chenopodium quinoa Willd.) Seeds in Comparison with Durum Wheat and Emmer

Antioxidant activity (AA) of quinoa (Chenopodium quinoa Willd.) seeds, as well as of durum wheat (Triticum turgidum L. ssp. durum Desf.) and of emmer (T. turgidum L. ssp. dicoccum Schübler) grains, was evaluated by studying hydrophilic (H), lipophilic (L), free-soluble (FSP) and insoluble-bound (IBP) phenolic extracts using the new lipoxygenase/4-nitroso-N,N-dimethylaniline (LOX/RNO) method, able to simultaneously detect different antioxidant mechanisms, as well as using the Oxygen Radical Absorbance Capacity (ORAC) and the Trolox Equivalent Antioxidant Capacity (TEAC) assays, which measure the scavenging activity against peroxyl and ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)] radicals, respectively. The species under study were compared with respect to the sum of AA values of H, L and FSP extracts (AA(H+L+FSP)), containing freely solvent-soluble antioxidants, and AA values of IBP extracts (AA(IBP)), representing the phenolic fraction ester-linked to insoluble cell wall polymers. The LOX/RNO and ORAC methods measured in quinoa flour a remarkable AA(H+L+FSP) higher than durum wheat, although lower than emmer; according to the same assays, the IBP component of quinoa resulted less active than the durum wheat and emmer ones. The TEAC protocol also revealed a high AA(H+L+FSP) for quinoa. Interestingly, the ratio AA(H+L+FSP)/AA(H+L+FSP+IBP), as evaluated by the LOX/RNO and ORAC assays, resulted in quinoa higher than that of both durum wheat and emmer, and much higher than durum wheat, according to the TEAC protocol. This may suggest that antioxidants from quinoa seeds may be more readily accessible with respect to that of both the examined wheat species. Quinoa seeds may represent an excellent source of natural antioxidant compounds and, in particular, of the free-soluble antioxidant fraction. These compounds may improve nutritive and health-beneficial properties of quinoa-based gluten-free products, thus expanding interest for quinoa utilization from celiac patients to the general population.

CELL WALL DISASSEMBLY DURING ON-TREE MATURATION, RIPENING AND SENESCENCE OF 'SNOW QUEEN' NECTARINES

In order to monitor ripening-related cell wall modifications, samples of the 'Snow Queen' nectarine cultivar were picked periodically around the commercial harvest date. Yields of insoluble cell wall materials (CWM) in the flesh tissue decreased significantly during the sampling period in parallel to increased yields of the soluble fraction, indicative of a progressive depolymerisation and solubilisation of the cell wall components. Further fractionation of the CWM showed a strong correlation between flesh firmness and the content of covalently-bound pectins, which thus appeared a key factor determining this quality attribute in 'Snow Queen' nectarines. Although to a lesser extent, the content of matrix glycans was also well related to preservation of flesh firmness. In contrast, no softening-related changes were observed for pectins bound non-covalently to the cell wall, suggesting a minor role for these polymers in the softening process of this nectarine cultivar. Cell-wall-modifying enzyme activities considered herein (polygalacturonase, pectate lyase and endo-(1-4)-β-D-glucanase) were generally higher at earlier picking dates and decreased as softening was taking place. These activities alone were not able to explain the modifications observed in flesh softening and cell wall composition, thus suggesting the synergistic action of a number of enzymes in the ripening-related softening of 'Snow Queen' nectarines.

Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be triggered by increase of cytosolic Ca(2+). Entry of Ca(2+) through the Ca(2+) release-activated Ca(2+) (CRAC) channels is under powerful regulation by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, FcεRI-dependent degranulation is decreased in SGK1-deficient (sgk1(-/-)) MCs. The present study addressed whether SGK1 is required for actin cytoskeleton rearrangement in MCs and whether modulation of actin architecture could underlie decreased degranulation of sgk1(-/-) MCs. Confirming previous results, release of β-hexosaminidase reflecting FcεRI-dependent degranulation was impaired in sgk1(-/-) MCs compared with sgk1(+/+) MCs. When CRAC channels were inhibited by 2-aminoethoxydiphenyl borate (2-APB; 50 μM), MC degranulation was strongly decreased in both sgk1(+/+) and sgk1(-/-) MCs and the difference between genotypes was abolished. Moreover, degranulation was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin B) agents to a similar extent in sgk1(+/+) MCs and sgk1(-/-) MCs, implying a regulatory role of actin reorganization in this event. In line with this, measurements of monomeric (G) and filamentous (F) actin content by FACS analysis and Western blotting of detergent-soluble and -insoluble cell fractions indicated an increase of the G/F-actin ratio in sgk1(+/+) MCs but not in sgk1(-/-) MCs upon FcεRI ligation, an observation reflecting actin depolymerization. In sgk1(+/+) MCs, FcεRI-induced actin depolymerization was abolished by 2-APB. The observed actin reorganization was confirmed by confocal laser microscopic analysis. Our observations uncover SGK1-dependent Ca(2+) entry in mast cells as a novel mechanism regulating actin cytoskeleton.

A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.

Sensitive Luminometric Method for Protein Quantification in Bacterial Cell Lysate Based on Particle Adsorption and Dissociation of Chelated Europium

A sensitive and rapid assay for the quantification of proteins, based on sample protein adsorption to Eu(3+)-chelate-labeled nanoparticles, was developed. The lanthanide ion of the surface-conjugated Eu(3+) chelate is dissociated at a low pH, decreasing the luminescence signal. The increased concentration of the sample protein prevents dissociation of the chelate, leading to a high luminescence signal due to the nanoparticle-bound protein. The assay sensitivity for the quantification of proteins was 130 pg for bovine serum albumin (BSA), which is an improvement of nearly 100-fold from the most sensitive commercial methods. The average coefficient of variation for the assay of BSA was 8%. The protein-to-protein variability was sufficiently low; the signal values varied within a 28% coefficient of variation for nine different proteins. The developed method is relatively insensitive to the presence of contaminants, such as nonionic detergents commonly found in biological samples. The existing methods tested for the total protein quantification failed to measure protein concentration in the presence of bacterial cell lysate. The developed method quantified protein also in samples containing insoluble cell components reducing the need for additional centrifugal assay steps and making the concept highly attractive for routine laboratory work.

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein–cell wall interactions.

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.

Dynamic response of UV-absorbing compounds, quantum yield and the xanthophyll cycle to diel changes in UV-B and photosynthetic radiations in an aquatic liverwort

We studied the diel responses of the liverwort Jungermannia exsertifolia subsp. cordifolia to radiation changes under laboratory conditions. The samples were exposed to three radiation regimes: P (only PAR), PA (PAR + UV-A), and PAB (PAR + UV-A + UV-B). The day was divided in four periods: darkness, a first low-PAR period, the high-PAR plus UV period, and a second low-PAR period. After 15 days of culture, we measured photosynthetic pigments, chlorophyll fluorescence and UV-absorbing compounds in the four periods of the day on two consecutive days. With respect to UV-absorbing compounds, we analyzed their global amount (as the bulk UV absorbance of methanolic extracts) and the concentration of seven hydroxycinnamic acid derivatives, both in the soluble (mainly vacuolar) and insoluble (cell wall-bound) fractions of the plant extracts. PAB samples increased the bulk UV absorbance of the soluble and insoluble fractions, and the concentrations of p-coumaroylmalic acid in the soluble fraction and p-coumaric acid in the cell wall. Most of these variables showed significant diel changes and responded within a few hours to radiation changes (more strongly to UV-B), increasing at the end of the period of high-PAR plus UV. F v/ F m, Φ PSII, NPQ and the components of the xanthophyll cycle showed significant and quick diel changes in response to high PAR, UV-A and UV-B radiation, indicating dynamic photoinhibition and protection of PSII from excess radiation through the xanthophyll cycle. Thus, the liverwort showed a dynamic protection and acclimation capacity to the irradiance level and spectral characteristics of the radiation received.

Abstract LB-190: Novel cell cycle regulation through the WW-domain of the co-chaperone BAG3

Abstract The BAG family of anti-apoptotic co-chaperones regulates protein folding and aggregation during cellular stress. The C-terminal BAG domain directly binds the ATPase domain of Hsp70/Hsc70 and prevents the release of client proteins that are otherwise directed to the proteasome for degradation. BAG3 is a unique member of the BAG family that also contains an evolutionarily conserved N-terminal WW-domain with undefined function. We removed the BAG3 WW-domain and observed a marked change in cell shape and cell cycle progression. This led us to hypothesize the BAG3 WW-domain is critical in regulating cellular division through a cytoskeletal association in a cell cycle-dependent fashion. Immunoblot analysis of synchronized HeLa (p53-null) and U2OS (human osteosarcoma, wild-type p53) cell lysates revealed that BAG3 expression was significantly increased during S phase and precipitated in the insoluble cell fraction at the G2/M transition. Indirect immunofluorescence demonstrated that BAG3 colocalized with the Golgi apparatus in the perinuclear region throughout interphase, and with leading lamellipodia exclusively during G2 phase. Co-immunoprecipitation of BAG3 with F-actin confirmed association with microfilaments and was increased during G2 phase relative to asynchronous lysates. A GST-hBAG3-WW-domain recombinant protein pulldown confirmed a specific F-actin association, suggesting a unique BAG3-WW-domain interaction with the cytoskeleton. We also generated a series of N-terminal, V5 tag-labeled domain-deletion clones of BAG3 with the WW (ΔWW) domain removed. ΔWW-BAG3 cells contained degenerate F-actin organization, abnormal morphology, and loss of BAG3 in lamellipodia. A 3.2-fold increase in DNA content of ΔWW-BAG3 cells was shown by flow cytometry, relative to empty vector (pCI Neo) and BAG3 overexpressing (FL-BAG3) transfected controls (p&amp;lt;0.001, n=3). Furthermore, centrosome amplification was increased in asynchronous cells: 2.43 ± 0.12/ΔWW-BAG3 cell, 1.86 ± 0.06/pCI Neo cell, and 1.79 ± 0.04/FL-BAG3 (p&amp;lt;0.05, n=4). Cell division in real time was followed using GFP-histone 2B-tagged live cell imaging. We confirmed polyploidy development and absence of cytokinesis in ΔWW-BAG3 cells, and also observed mitotic delay relative to pCI Neo (1260 min. vs. 110 min.) and FL-BAG3 cells (vs. 90 min., n=2). These data indicate a novel requirement for BAG3 for normal mitotic progression via BAG3 WW-domain association with the actin cytoskeleton. To our knowledge, this is the first reported activity of differential co-chaperone protein behavior based on cell cycle phase, independent of p53 status. These data identify a novel regulatory point of G2/M progression and suggest the WW-domain of BAG3 is a potential therapeutic target to deter cancer proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-190. doi:10.1158/1538-7445.AM2011-LB-190

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.

Cell wall polysaccharides of Chinese Wolfberry (Lycium barbarum): Part 1. Characterisation of soluble and insoluble polymer fractions

Water-soluble polysaccharides (WSP) and insoluble cell wall material (CWM) were isolated from Wolfberry fruit (Lycium barbarum). WSP were fractionated by treatment with a quaternary ammonium salt and anion-exchange chromatography. Pectic polysaccharides were major components but a glucan, xylan and arabinogalactan-proteins (AGP) were also present. CWM was fractionated into pectic and hemicellulosic polysaccharides by a sequential solvent extraction. The former were rhamnogalacturonans with varying degrees of branching of the backbone. A (4-O-methyl-glucurono)xylan was the predominant hemicellulose. The additional presence of xyloglucan and a mannan-based polymer, strongly attached to the cellulose fibrils was indicated. Of the 6.8% total polysaccharide content of Wolfberry fruit, 1.2% was readily water soluble, 5.6% insoluble and 0.3% was readily soluble AGP.

Kinetics fermentation and gas production of the neutral detergent-soluble fraction of fresh forage, silage and hay of Avena sativa

Neutral detergent treatment to evaluate the structural polysaccharides (NDF) removes soluble sugars, pectin substances, starch and the non-carbohydrate fraction (Van Soest, 1991). Despite the widely recognized importance of neutral detergent-soluble carbohydrates (NDS) in the early stages of forage digestion, little is known about their digestion kinetics because most in vitro methods have studied the disappearance of insoluble cell wall components. However, knowledge of the degradation characteristics of both fractions is very important to formulate balanced diets for high-yielding animals. Using the in vitro gas production system........

Substrate-binding properties of the family 54 module of Clostridium thermocellum Lic16A laminarinase

Endo-β-1.3–1.4-glucanase Lic16A of the moderate thermophilic anaerobe Clostridium thermocellum has a complex multimodular structure. In addition to the catalytic module, Lic16A contains eight auxiliary modules, five of which are substrate-binding modules. The new family 54 substrate-binding module CBM54 (25.2 kDa), which is at the N terminus of the enzyme, has a cleavage site in its N-terminal part, and its cleavage yields a shortened module CBM54C (17.2 kDa) in vivo and in vitro. CBM54C was cloned in Escherichia coli and purified to electrophoretic homogeneity. The binding constants of CBM54C to xylan, chitin, insoluble yeast cell wall β-glucan, and bacterial crystalline cellulose were of the same order of magnitude as for CBM54. However, CBM54C did not bind pustulan, avicel, and chitosan, in contrast to CBM54. Calcium ions restored the ability of CBM54C to bind these three carbohydrates. CBM54 substrate binding promiscuity suggested multiple binding sites, some of them being Ca2+ dependent. The Ca2+-independent sites for avicel, pustulan and chitosan were localized to the spontaneously split-off N-terminal part (8 kDa) of CBM54. The arrangement of Ca2+-dependent and Ca2+-independent binding sites for various substrates was suggested.

Phenolics and Antioxidant Properties of Fruit Pulp and Cell Wall Fractions of Postharvest Banana (Musa acuminata Juss.) Cultivars

Banana fruits are important foods, but there have been very few studies evaluating the phenolics associated with their cell walls. In the present study, (+) catechin, gallocatechin, and (-) epicatechin, as well as condensed tannins, were detected in the soluble extract of the fruit pulp; neither soluble anthocyanidins nor anthocyanins were present. In the soluble cell wall fraction, two hydroxycinnamic acid derivatives were predominant, whereas in the insoluble cell wall fraction, the anthocyanidin delphinidin, which is reported in banana cell walls for the first time, was predominant. Cell wall fractions showed remarkable antioxidant capacity, especially after acid and enzymatic hydrolysis, which was correlated with the total phenolic content released after the hydrolysis of the water-insoluble polymer, but not for the posthydrolysis water-soluble polymer. The acid hydrolysis released various monosaccharides, whereas enzymatic hydrolysis released one peak of oligosaccharides. These results indicate that banana cell walls could be a suitable source of natural antioxidants and that they could be bioaccessible in the human gut.

Analysis of Detergent-Insoluble and Whole Cell Lysate Fractions of Resting Neutrophils Using High-Resolution Mass Spectrometry

Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass

Insoluble cell wall material was prepared from the skin and flesh of commercially ripe Vitis vinifera L. cv. Shiraz berries and then combined in suspension with preveraison skin and seed proanthocyanidin containing solutions. Analysis of proanthocyanidins before and after fining with cell wall material by phloroglucinolysis provided information on recovery by mass, subunit composition, and mean degree of polymerization, whereas proanthocyanidin molecular mass distribution was determined by gel permeation chromatography. Cell wall material from flesh showed the highest affinity for proanthocyanidin, binding up to 47% and 57% w/w of total seed and skin proanthocyanidin respectively. Comparison of the molecular mass distribution of skin or seed proanthocyanidin before and after fining indicated that affinity of cell walls for proanthocyanidin increased with increasing proanthocyanidin molecular mass. Initial results of subunit composition of skin and seed proanthocyanidin mixtures following fining with cell wall material showed that the % galloylation decreased, suggesting a preference for seed-derived proanthocyanidins. Subsequent experiments suggest that fining with insoluble cell wall material is size-based and does not have a specific affinity for seed-derived proanthocyanidins.

De-Ubiquitinase Inhibition by WP1130 Induces Formation of Aggresomes, Engages Autophagy and Activates Apoptosis in B-Cell Malignancies.

Abstract 3769Poster Board III-705Nearly all protein in mammalian cells is subject to regulation by ubiquitination and it is becoming clear that modification of this process may have therapeutic activity in several diseases, including cancer. Bortezomib and Hsp90 inhibitors affect protein behavior through ubiquitination and have therapeutic activity in specific cancers. These observations suggest that additional molecules that alter protein ubiquitination through a distinct mechanism may have therapeutic value. Previously we described a small molecule, WP1130, that inhibited cytokine and Bcr-Abl mediated signaling through a unique mechanism (Blood. 2007;109(8):3470-8, Cancer Res. 2007;67(8):3912-8). In this study we examined the mechanism of WP1130 action. Like bortezomib, WP1130 induced rapid, time and dose-dependent protein ubiquitination in treated cells. However unlike bortezomib, WP1130 treatment resulted in accumulation of ubiquitinated proteins in the detergent insoluble cell fraction. Sub-cellular fractionation studies revealed deposition of ubiquitinated proteins into the cytoskeletal-enriched fraction of intra-cellular structures called aggresomes. Since proteosome inhibition also results in aggresome formation, confocal microscopic examination of WP1130 treated cells was performed which demonstrated formation of peri-nuclear aggresomes containing ubiquitinated proteins, in complex with heat shock chaperones and other aggresome markers such as HDAC6 and γ-tubulin. However, WP1130 did not directly or indirectly inhibit the 20S proteasome, did not disrupt HSP90 activity or induce oxidative stress, distinguishing its mechanism from other agents. Since aggresomes also acts as disposal grounds for accumulated insoluble protein aggregates through autophagy, we assessed whether autophagy was engaged as a consequence of aggresome formation in WP1130 treated cells. We observed activation of the autophagy pathway, evident by the rapid conversion of LC3-I to LC3-II and decrease in p62/SQSTM1 protein levels. We also compared WP1130 to its parental compound, AG490, and found no changes in ubiquitination or an induction of autophagy with AG490. Since autophagy and aggresome formation are known to be cytoprotective measures initiated by cells to cope with unfavorable growth conditions, inhibition of autophagy may enhance cytotoxicity associated with WP1130. Co-treatment of cells with WP1130 and wortmannin, a known inhibitor of autophagy, resulted in a rapid increase of caspase-3 activity, culminating in enhanced cell death. Biochemical evidence supports a role for de-ubiquitinase (DUB) inhibition in WP1130 treated cells as a mechanism of enhanced resident ubiquitination of cellular proteins. Lysates from WP1130 treated cells displayed time- and concentration-dependent decreased DUB activity. These results suggest that WP1130 initiates rapid and robust protein ubiquitination through suppression of cellular DUB activity to initiate an alternate pathway to control protein behavior and induce apoptosis in specific B-cell malignancies which are highly sensitive to WP1130 and other ubiquitin content modifying agents. Disclosures:No relevant conflicts of interest to declare.

Biotransformation of β-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene beta-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with beta-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform beta-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either beta-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on beta-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a beta-myrcene degradation pathway in this organism.

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

Plasmid DNA production combining antibiotic‐free selection, inducible high yield fermentation, and novel autolytic purification

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.