Bulk Proteins Research Articles

Relating algal-derived extracellular and intracellular dissolved organic nitrogen with nitrogenous disinfection by-product formation

The dissolved organic nitrogen (DON) pool from algal-derived extracellular and intracellular organic matter (EOM and IOM) comprises proteins, peptides, free amino acids and carbohydrates, of which, proteins can contribute up to 100% of the DON. Previous reports of algal-derived DON character have focused on bulk properties including concentration, molecular weight and hydrophobicity. However, these can be similar between algal species and between the EOM and IOM even when the inherent molecular structures vary. A focus on bulk character presents challenges to the research on algal-derived nitrogenous-disinfection by-product (N-DBP) formation as N-DBP formation is sensitive to the changes in molecular structure. Hence, the main aim of this study was to characterize algal EOM and IOM-derived DON, specifically proteinaceous-DON, using a combination of bulk and molecular characterization techniques to enable a more detailed exploration of the relationship between the character of algal-derived proteins and the N-DBP formation potential. DON from the EOM and IOM of four commonly found algae and cyanobacteria in natural waters were evaluated, namely Chlorella vulgaris, Microcystis aeruginosa, Dolichospermum circinale, and Cylindrospermopsis raciborskii. It was observed that 77-96% of total DON in all EOM and IOM samples was of proteinaceous origin. In the proteins, DON concentrations were highest in the high molecular weight fraction of IOM-derived bulk proteins (0.13–0.75 mg N L−1) and low to medium molecular weight fraction of EOM-derived bulk proteins (0.15–0.63 mg N L−1) in all species. Similar observations were also made via sodium dodecyl sulphate polyacrylamide gel electrophoresis and liquid chromatography-high resolution mass spectrometry. Solid-state 15N nuclear magnetic resonance (NMR) spectroscopy of the EOM and IOM revealed the existence of common aliphatic and heterocyclic N-groups in all samples, including a dominant 2° amide peak. Species dependent variability was also observed in the spectra, particularly in the EOM; e.g. nitro signals were found only in the Cylindrospermopsis raciborskii EOM. Dichloroacetonitrile (DCAN) and N-nitrosamine concentrations from the EOM of the species evaluated in this study were lower than the guideline limits set by regulatory agencies. It is proposed that the dominant 2° amide in all samples decreased N-DBP formation upon chlorination. For chloramination, the presence of nitro groups and aliphatic and heterocyclic N-DBP precursors could cause variable N-nitrosamine formation. Compared to non-algal impacted waters, algae-laden waters are characterised by low organic carbon: organic nitrogen ratios of ∼7–14 and elevated DON and protein concentrations. Hence, relying only on bulk characterization increases the perceived risk of N-DBP formation from algae-laden waters.

One-pot synthesis of high-concentration mixed-shell polymeric micelles as nanochaperones for the renaturation of bulk proteins

A one-pot synthesis of high-concentration mixed-shell polymeric micelles and synthetic nanoparticles can be used to assist the refolding of bulk denatured proteins and stabilize native proteins for long-term storage.

Characterizing extracellular vesicles from cerebrospinal fluid by a novel size exclusion chromatography method

AbstractBackgroundExtracellular vesicles (EVs) have recently been considered as a potential biomarker source for a variety of diseases, including neurodegenerative disorders. EVs are important mediators of intercellular communication due to their capacity to transfer genetic material, lipids and proteins. By means of their communication role, interesting biomarkers are often enriched in EVs compared to total biofluid. In order to isolate EVs from a biofluid, like cerebrospinal fluid (CSF), a variety of isolation methods are available of which each methods shows different results, and, to date, there is no golden standard. Our study evaluates the use of size exclusion chromatography (SEC) by the SmartSEC HT kit for EV isolation from CSF.MethodEVs were obtained from 500µL CSF samples, isolated by SmartSEC HT. SmartSEC traps contaminants, like bulk proteins, into the resin beads and subsequently elutes EVs. To evaluate this isolation method, various characterisation analyses were run. To detect cargo or surface proteïns, analyses on the ExoView R100, Surface Plasmon Resonance imaging (SPRi) and LC‐MS based proteomics analysis were performed. Concentration, and size of the vesicles were evaluated by use of transmission electron microscopy (TEM), and fluorescence nanotracking analysis (Zetaview).ResultBy means of the ExoView, tetraspanins such as CD81 and CD9 were measured on the surface of the EVs. Other EV‐specific proteins, e.g. lactadherin, flotillin‐1, and HSP70, were detected by SPRI. Untargeted protein analysis revealed the cargo proteins. TEM images visualized the presence of EVs. Particle size and concentration was calculated by nanotracking analysis, and more specifically the particles with a lipid membrane, like EVs, with fluorescence nanotracking analysis.ConclusionAs EVs hold a lot of promise in the biomarker field, a standard high‐performance isolation method ‐ regarding purity and yield – is essential. From our data, SmartSEC HT is suggested to be an efficient size exclusion chromatography EV isolation method for low volume CSF samples, especially for high throughput, since samples are loaded on a 96‐well plate.

Proteogenomic Characterization of the Cement and Adhesive Gland of the Pelagic Gooseneck Barnacle Lepas anatifera.

We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.

Outer membrane proteins (OMPs) profiling of a field isolate of Salmonella Typhimurium: Isolation and its characterization

Salmonella Typhimurium is a significant zoonotic pathogen affecting human and animal health. In the present study, a S. Typhimurium isolate, previously isolated from poultry samples was revived and re-confirmed as S. Typhimurium by isolation in Hektoen enteric agar followed by molecular detection. PCR with the stn gene, his J gene and Typh gene primers revealed the specific amplicon of size 617 bp, 496 bp and 402 bp respectively. Antibiotic sensitivity pattern of the isolate was also determined. Isolate was then cultured in LB broth in bulk and outer membrane proteins (OMPs) were isolated by ultracentrifugation. The OMPs were successfully isolated and confirmed by SDS-PAGE analysis. The band pattern revealed nine major bands ranging from 11-90 kDa.

Protein based evaluation of meat species by using laser induced breakdown spectroscopy

Meat adulteration through partial substitution with cheaper species or mislabeling causes significant problems in terms of health, religious beliefs, economy, and product quality. Therefore, identification of meat species is crucial for monitoring and prevention of meat fraud. In the present study, protein based laser induced breakdown spectroscopy method was developed for the first time to identify three meat species (beef, chicken and pork) by using bulk proteins and protein fractions, namely actin and myosin. LIBS spectra were evaluated with principal component analysis for clustering pattern of meat species, and partial least square analysis was performed to determine adulteration ratio. In PLS analysis, limit of detection (LOD) values for beef adulteration with chicken and pork meat were calculated as 2.84% and 3.89% by using bulk proteins, respectively.

Adaptive methodology to determine hydrophobicity of nanomaterials in situ.

The hydrophobicity of nanoparticles (NPs) is a key property determining environmental fate, biological partitioning and toxicity. However, methods to characterize surface hydrophobicity are not uniformly applied to NPs and cannot quantify surface changes in complex environments. Existing methods designed to evaluate the hydrophobicity of bulk solids, chemicals, and proteins have significant limitations when applied to NPs. In this study, we modified and evaluated two methods to determine the hydrophobicity of NPs, hydrophobic interaction chromatography (HIC) and dye adsorption, and compared them to the standard octanol-water partitioning protocol for chemicals. Gold, copper oxide, silica, and amine-functionalized silica NPs were used to evaluate methods based on their applicability to NPs that agglomerate and have surface coatings. The octanol water partitioning and HIC methods both measured Au NPs as hydrophilic, but despite having a small size and stable suspension, NPs could not be fully recovered from the HIC column. For the dye adsorption method, hydrophobic (Rose Bengal) and hydrophilic (Nile Blue) dyes were adsorbed to the NP surface, and linear isotherm parameters were used as a metric for hydrophobicity. CuO was determined to be slightly hydrophilic, while SiO2 was hydrophilic and Ami-SiO2 was hydrophobic. The advantages and limitations of each method are discussed, and the dye adsorption method is recommended as the most suitable for application across broad classes of nanomaterials. The dye assay method was further used to measure changes in the surface hydrophobicity of TiO2 NPs after being suspended in natural water collected from the Alsea Rivers watershed in Oregon. TiO2 NPs adsorbed Rose Bengal when suspended in ultrapure water, but adsorbed Nile Blue after being incubated in natural water samples, demonstrating a shift from hydrophobic to hydrophilic properties on the outer surface. The dye adsorption method can be applied to characterize surface hydrophobicity of NPs and quantify environmental transformations, potentially improving environmental fate models.

Natural 15N abundance in specific amino acids indicates associations between transamination rates and residual feed intake in beef cattle.

Improving the ability of animals to convert feed resources into food for humans is needed for more sustainable livestock systems. Genetic selection for animals eating less while maintaining their performance (i.e., low residual feed intake [RFI]) appears a smart strategy but its effectiveness relies on high-throughput animal phenotyping. Here, we explored plasma nitrogen (N) isotope ratios in an attempt to identify easily superior young bulls in terms of RFI. For this, 48 Charolais young bulls fed two contrasting diets (corn vs. grass silage diets) were selected from a larger population as extreme RFI animals (24 low-RFI vs. 24 high-RFI) and their plasma analyzed for natural 15N abundance (δ15N) in the whole protein (bulk protein) and in the individual protein-bound amino acids (PbAA). For the first time, we showed that the δ 15N in plasma bulk protein differed (P = 0.007) between efficient (low-RFI) and inefficient (high-RFI) cattle regardless of diet. Furthermore, most analyzed PbAA followed the same trend as the bulk protein, with lower (P < 0.05) δ 15N values in more efficient (low-RFI) compared with less efficient (high-RFI) cattle, again regardless of diet. The only three exceptions were Phe, Met, and Lys (P > 0.05) for which the first metabolic reaction before being catabolized does not involve transamination, a pathway known naturally to enrich AAs in 15N. The contrasted isotopic signatures across RFI groups only in those PbAA undergoing transamination are interpreted as differences in transamination rates and N-use efficiency between low- and high-RFI phenotypes. Natural isotopic N signatures in bulk proteins and specific PbAA can be proposed as biomarkers of RFI in growing beef cattle fed different diets. However, the current study cannot delineate whether this effect only occurs post-absorption or to some extent also in the rumen. Our data support the conclusion that most efficient cattle in terms of RFI upregulate N conservation mechanisms compared with less efficient cattle and justify future research on this topic.

Sustainable extraction of valuable components from Spirulina assisted by pulsed electric fields technology

The study investigated the potential of pulsed electric fields (PEF) technology application for the improvement of cell disintegration and subsequent extraction/fractionation of cyanobacteria Arthrospira maxima into valuable components (phycocyanin, proteins, oils and carbohydrates). Pulsed electric fields were applied to fresh A. maxima suspension, dried powder and dried sticks in combination with water as extraction solvent and freeze-thawing. Pulsed electric fields application for cell disruption reached approximately 90% increase of C-phycocyanin extraction compared to bead milling. Obtained fractions of phycocyanin and bulk proteins were also of higher purity and had twice lower environmental impact than similar fractions obtained without Pulsed electric fields treatment. Extracts with improved purity can be directly applied in pharma and food industry without any further processing and purification steps.

An Open Source Platform for Continuum Simulations of Biological Membranes

Biological membranes, comprised of lipids and proteins, make up the boundary of the cell, as well as the boundaries of internal organelles such as the nucleus, endoplasmic reticulum, and Golgi complex. The mechanics and non-equilibrium thermodynamics of lipid membranes in biology are complex but play a pivotal role in many cellular processes such as endocytosis. Lipid membranes can (1) take shapes of arbitrary topology which (2) deform under large deformations resulting in different morphologies and (3) show significant surface flows. Here, we built upon a theoretical model that captures these three essential phenomena. Analytical solutions to the theoretical model do not generally exist and hence, computational methods are required. We developed an Arbitrary-Lagrangian-Eulerian theory and a corresponding Finite Elements (ALE-FE) Method that allows for arbitrarily deforming surfaces with in-plane surface flows. An important challenge in developing ALE-FE methods is the requirement of C1-continuous surface representations. Therefore, a novel meshing technique, based on subdivision surfaces inspired by computer graphics, is developed. We combined these two tools---an ALE-FE framework and a corresponding meshing technique---in the first open-source platform for continuum simulations of biological membranes. For the first time, this enables scientists to approach a manifold of research problems related to the mechanics and non-equilibrium thermodynamics of lipid membranes. In particular, this open-source platform aims at facilitating the exchange between experiments and theory. Our presentation will be complemented by fascinating examples, including mechanical instabilities of lipid membranes.The open-source platform is amenable to the extension to other non-equilibrium thermodynamics phenomena including surface diffusion, chemical reactions involving bulk proteins, and interactions between curved and deforming surfaces with bulk flows. Furthermore, the software can be extended to other materials such as elastic sheets.

New Class of Crosslinker-Free Nanofiber Biomaterials from Hydra Nematocyst Proteins

Nematocysts, the stinging organelles of cnidarians, have remarkable mechanical properties. Hydra nematocyst capsules undergo volume changes of 50% during their explosive exocytosis and withstand osmotic pressures of beyond 100 bar. Recently, two novel protein components building up the nematocyst capsule wall in Hydra were identified. The cnidarian proline-rich protein 1 (CPP-1) characterized by a “rigid” polyproline motif and the elastic Cnidoin possessing a silk-like domain were shown to be part of the capsule structure via short cysteine-rich domains that spontaneously crosslink the proteins via disulfide bonds. In this study, recombinant Cnidoin and CPP-1 are expressed in E. coli and the elastic modulus of spontaneously crosslinked bulk proteins is compared with that of isolated nematocysts. For the fabrication of uniform protein nanofibers by electrospinning, the preparative conditions are systematically optimized. Both fibers remain stable even after rigorous washing and immersion into bulk water owing to the simultaneous crosslinking of cysteine-rich domains. This makes our nanofibers clearly different from other protein nanofibers that are not stable without chemical crosslinkers. Following the quantitative assessment of mechanical properties, the potential of Cnidoin and CPP-1 nanofibers is examined towards the maintenance of human mesenchymal stem cells.

Accessibility of transglutaminase to induce protein crosslinking in gelled food matrices - Influence of network structure

Microbial transglutaminase (mTG) catalyzes the crosslinking of bulk and interfacial proteins, thereby influencing the physical properties of food dispersions. However, little is known about the impact of the spatial distribution of proteins in a gelled matrix on the enzymatic crosslinking. In this study, free and incorporated emulsions were subjected to mTG to induce crosslinking of emulsion interfaces. First, simple caseinate-stabilized emulsions were fabricated by high shear blending (20,000 rpm, 6 min). Second, emulsion samples were embedded into hydrogel beads using an injection technique. Different alginate (0.5–1.5%) and CaCl2 levels (50–500 mM) were used to modulate the hydrogel pore size and number of junction zones. Third, free and incorporated emulsions were mixed with mTG to investigate the diffusion behavior of the enzyme. Results showed that mTG diffused into the beads to an extent of more than 50%. A delay in ammonia release was observed when emulsions were incorporated into hydrogel beads, whereas protein in free emulsions was instantly crosslinked after mTG was added. These results illustrate that the spatial make-up of biopolymers in gelled matrices play a key role on the enzyme accessibility.

Abstract 3694: Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein

Abstract Goniothalamin (GTN) is a secondary metabolite styryl lactone isolated from several species of the tropical medicinal tree Goniothalamus. GTN has been shown to be cytotoxic and induce apoptosis in many cancer cell lines. This study sought to define the molecular basis underlying the antiproliferative actions of GTN. We synthesized the R and S enantiomers of GTN and found the R form to be more cytotoxic against a panel of breast cancer and lung cancer cell lines. The IC50 of GTN against breast cancer cell lines was in the range of 10-20 μM and interestingly, the SKBR3 cells, which harbor a R175H mutation in p53 were more sensitive to the drug. In contrast, the normal breast epithelial MCF10A cells were not killed by GTN, and N-acetylcysteine prevented cell-killing, indicating the ROS involvement. Indeed, the flow cytometry and cell staining with DCF-DA showed high levels of ROS generation, and this was accompanied by significant S-glutathionylation of bulk proteins. Other studies showed that GTN forms conjugate with glutathione with ease and deplete GSH levels in cells. Because p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by GTN may affect the structure of the R175H mutant p53 protein, and account for greater cytotoxicity. We also engineered the p53-null H1299 lung cancer cells to stably express the R175H mutant protein by lentiviral technology as an isogenic model. The conformation-specific antibodies for p53, namely Pab1620 that recognize the wt-p53 and pab420 that detects the mutant p53 were used for validating the p53 restoration. Immunoprecipitation and immunostaining using confocal microscopy showed that GTN treatment of R175H p53-containing cells results in a reciprocal loss of mutant protein and increase of wt-like protein. Further, the EMSA revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes. The changes were associated with a G2/M arrest and significant apoptosis. Increased levels of apoptotic markers suggested a priming action of GTN on cell death pathways. GTN also suppressed the SKBR3 cell migration and invasion at 5-10 μM. In SKBR3 xenografts developed in nude mice, there was a marked tumor growth delay with either GTN alone or in combination with cisplatin. Our results shed light on the multiple mechanisms, including glutathione depletion, generation of redox imbalance, protein glutathionylation and p53 reactivation in the GTN cytotoxicity. We suggest that GTN-induced oxidative milieu facilitates a functional restoration of the mutant p53 through a thiolation of the redox-sensitive cysteines present in the DNA-binding domain. Our study will help to establish redox-perturbation as a paradigm for reactivation of the defective tumor suppressor (supported by a CPRIT grant [RP130266] to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Debasish Basak, Kalkunte S. Srivenugopal. Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3694.

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones.

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO, NO, Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.

Abstract 155: Expressions of autophagy-related proteins positively correlate with infiltration of immune cells and disease progression in oral squamous cell carcinoma

Abstract Introduction: Autophagy is an endogenous process that degrades bulk proteins and organelles to maintain energy homeostasis under cellular stress and nutrient starvation. It has been still unclear whether autophagy promotes or suppresses tumor progression in various cancer patients. To date, several studies have shown that autophagy-related molecules such as LC3 and Beclin-1 play important roles in tumor progression; however, few studies regarding immunological significance of these autophagy components in tumor microenvironment exist. The objective of this study is to clarify association between the expression of autophagy-related proteins and immune infiltrates by immunohistochemistry (IHC). Patients and methods: Expression of two representative autophagy-associated molecules, LC3 and Beclin-1, and CD1a (dendritic cell), CD3 (T cell) and CD56 (NK cell) cells were examined by IHC in 74 patients with oral squamous cell carcinoma (OSCC). HLA class I heavy chain, HLA class II, Ki-67 and p53 were also stained and assessed. Two regions, central site and peripheral site in tumor tissues were evaluated, separately. Moreover, the association with various clinicopathological parameters including progression free and overall survival was evaluated. Results: LC3 and Beclin-1 were accumulated in both 27 of 74 cases (36.5%) OCCC examined. LC3 expression in peripheral margin was significantly associated with increasing dendritic cell (DC) and T cell infiltration. Interestingly, peripheral Beclin-1 expression showed statistically significant relation to increasing HLA class II expression on tumor cells. On the other hand, LC3 and Beclin-1 significantly correlates with several unfavorable clinicopathological parameters including short overall survival. In addition, LC3 and Beclin-1 expression in OSCC significantly related to lymphatic invasion by tumor cells. Conclusion: Significant association between LC3 and DC/T cell infiltration suggests that autophagy is likely to actively participate in immunological phenomena within tumor microenvironment. Furthermore, peripheral Beclin-1 may contribute to antigen presentation by tumor cells, while these two autophagy components also relates to unfavorable prognosis. Taken together, autophagy seems to promote immunogenicity of cancer cells, whereas it facilitates tumor progression. Citation Format: Koichi Sakakura, Hideyuki Takahashi, Kyoichi Kaira, Minoru Toyoda, Tetsunari Oyama, Kazuaki Chikamatsu. Expressions of autophagy-related proteins positively correlate with infiltration of immune cells and disease progression in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 155. doi:10.1158/1538-7445.AM2014-155

Degradation of NF-κB, p53 and other regulatory redox-sensitive proteins by thiol-conjugating and -nitrosylating drugs in human tumor cells

The ionized cysteines present on the surfaces of many redox-sensitive proteins play functionally essential roles and are readily targeted by the reactive oxygen and reactive nitrogen species. Using disulfiram (DSF) and nitroaspirin (NCX4016) as the model compounds that mediate thiol-conjugating and nitrosylating reactions, respectively, we investigated the fate of p53, nuclear factor-kappaB (NF-κB) and other redox-responsive proteins following the exposure of human cancer cell lines to the drugs. Both drugs induced glutathionylation of bulk proteins in tumor cells and cell-free extracts. A prominent finding of this study was a time- and dose-dependent degradation of the redox-regulated proteins after brief treatments of tumor cells with DSF or NCX4016. DSF and copper-chelated DSF at concentrations of 50-200 µM induced the disappearance of wild-type p53, mutant p53, NF-κB subunit p50 and the ubiquitin-activating enzyme E1 (UBE1) in tumor cell lines. DSF also induced the glutathionylation of p53. The recombinant p53 protein modified by DSF was preferentially degraded by rabbit reticulocyte lysates. The proteasome inhibitor PS341 curtailed the DSF-induced degradation of p53 in HCT116 cells. Further, the NCX4016 induced a dose-dependent disappearance of the UBE1 and NF-κB p50 proteins in cell lines, besides a time-dependent degradation of aldehyde dehydrogenase in mouse liver after a single injection of 150 mg/kg. The loss of p53 and NF-kB proteins correlated with decreases in their specific binding to DNA. Our results demonstrate the hitherto unrecognized ability of the non-toxic thiolating and nitrosylating agents to degrade regulatory proteins and highlight the exploitable therapeutic benefits.

Abstract 3744: Brain's High Constitutive Background Autophagy Level Is Disrupted by Stroke Injury and Post-stroke Pharmacological Induction of Autophagy Reduces Lesion Size

Background: Autophagy is a highly regulated and dynamic process that allows cells to recycle aging organelles, bulk proteins and lipids for nutritional reuse. Autophagy occurs in all cell types and body tissues and is induced by oxidative stress or lack of nutrients to promote survival. Excessive autophagy, however, can lead to cell death, including apoptotic (PCD type I) and autophagic cell death (PCD Type II). Recent literature reports that autophagy is disregulated during stroke and other brain injuries. We hypothesized that the normally high innate level of autophagy in the brain undergoes aberrant regulation during stroke injury and this aberrant regulation correlates to changes in stroke lesion size. Methods: We induced ischemia in C57BL/6J mice using a permanent MCA ligation model, with sacrifice 48 hours after stroke. For time course analysis mice were sacrificed at 3, 6, 12, 24, 48, or 72 hours after stroke. Mice were treated i.p. with chloroquine (30, 60, or 90 mg/K/d for 2 days), an autophagy inhibitor, rapamycin (1.25 or 2.5 mg/K/d for 2 days), an autophagy activator via inhibition of mTOR, or treated with vehicle (saline or 10% DMSO in 100 ul). The autophagy markers LC3, p62 and Beclin-1 were analyzed by Western blot and immunohistochemistry and infarct size was measured by TTC staining. Results: Brain showed consistently higher baseline levels of LC3II than other tissues. Chloroquine and rapamycin both reduced lesion size, the former between 28 &amp; 34% and the later, in a dose dependent fashion, between 60 and 65%. Beclin-1 is decreased in the infarct side of the brain and treatment with rapamycin enhanced this depletion while treatment with chloroquine increased Beclin-1 levels. LC3II is decreased in the infarct side and does not increase with chloroquine treatment on the infarct side, suggesting that stroke injury may inhibit the high innate baseline level of autophagy in the brain. Rapamycin led to increased LC3II and p62, consistent with increased induction of autophagy. Time course analysis of stroked brains showed that LC3II and Beclin-1 are initially decreased at 3 to 6 hours post injury, then increased above non-injured between 12 and 24 hours before it falls below baseline at 48 to 72 hours. Conclusions: 1) Stroke appears to inhibit the high innate baseline levels of autophagy in the brain after the injury. 2) Since chloroquine and rapamycin both decreased lesion size, this suggests cross-talk between autophagy and other programmed cell death pathways and/or direct effects of the drugs on these and other pathways. 3) The rapid decrease in autophagy markers post injury indicates that modulation of autophagy may offer a new therapeutic avenue for stroke.

A liquid chromatography–tandem mass spectrometry method for the detection of economically motivated adulteration in protein-containing foods

A new analytical method was developed to determine the presence of six (6) compounds with the potential to be used in economic adulteration to enhance the nitrogen content in milk products and bulk proteins. Residues were extracted from the matrix with 2% formic acid, after which acetonitrile (ACN) was added to induce precipitation of the proteins. Extracts were analyzed by liquid chromatography using a ZIC-HILIC column with tandem mass spectrometry (LC-MS/MS) using electrospray ionization (ESI). Single-laboratory method validation data was collected in six matrices fortified at concentrations down to 1.0 μg/g (ppm). Average recoveries and average relative standard deviations (RSD) using spiked matrix calibration standard curves were the following: cyromazine (CY) 95.9% (7.5% RSD), dicyandiamide (DC) 98.1% (5.6% RSD), urea 102.5% (8.6% RSD), biuret (BU) 97.2% (6.6% RSD), triuret (TU) 97.7% (5.7% RSD), and amidinourea (AU) 93.4% (7.4% RSD). This method provides a rapid and effective approach to proactively combat economically motivated adulteration in protein-containing products.

Staphylococcus aureus ClpC is involved in protection of carbon-metabolizing enzymes from carbonylation during stationary growth phase

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Among them, ClpC, belonging to the Hsp100/Clp ATPase family, seems to play an important role. For instance, we previously demonstrated that a functional clpC deletion resulted in enhanced survival in the late stationary phase (death phase period) compared to the parental S. aureus strain. However, the mechanisms for the enhanced survival of a S. aureus clpC mutant during the death phase period are still elusive. In Escherichia coli, among the factors that might lead to bacterial cell death during stationary phase, the amount of protein aggregates and/or oxidized proteins appears to be of major importance. Thus, in the present study, we have evaluated protein aggregates and carbonylated protein (as a marker of protein oxidation) contents both in the wild type and in an S. aureus clpC mutant during the exponential growth phase and the death phase. Whereas at all time points the tested clpC mutant exhibits the same amount of protein aggregates as the WT strain, the total amount of carbonylated proteins appears to be lower in the clpC mutant. Moreover, we observed that at the entrance of the death phase carbon-metabolizing enzymes [such as the TCA cycle enzymes Mqo2 (malate: quinone oxidoreductase) and FumC/CitG (fumarate hydratase)] albeit not the bulk proteins are carbonylated to a larger extent in the clpC mutant. Reduced activity of the TCA cycle due to specific carbonylation of these proteins will result in a decrease of endogenous oxidative stress which in turn might confer enhanced survival of the clpC mutant during the death phase period thus contributing to bacterial longevity and chronic infection.

Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product

Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient from a formulated biological drug product in a manner that is consequently sufficient for its use in comparability studies.