Development of cancer biomarker heat shock protein 90α certified reference material using two different isotope dilution mass spectrometry techniques.

Heat shock protein 90α (HSP90α) has been regarded as an important indicator for judging tumor metastasis and prognosis due to its significant upregulation in various tumors. Therefore, the accurate quantification of HSP90α is of great significance for clinical diagnosis and therapy of cancers. However, the lack of HSP90α certified reference material (CRM) leads to the accuracy and consistency of quantification methods not being effectively evaluated. Besides, quantitative results without traceability make comparisons between different studies difficult. In this study, an HSP90α solution CRM was developed from the recombinant protein raw material. The recombinant protein is a dimer, and the purity of the CRM candidate reached 96.71%. Both amino acid analysis-isotope dilution mass spectrometry (AAA-IDMS) and unique peptide analysis-isotope dilution mass spectrometry (UPA-IDMS) were performed to measure the content of HSP90α in the solution CRM candidate, and the certified value was assessed to be 66.2 ± 8.8 µg/g. Good homogeneity of the CRM was identified, and the stability examination suggested that the CRM was stable for at least 4 months at - 80 °C and for 7 days at 4 °C. With traceability to SI unit (kg), this CRM has potential to help establish a metrological traceability chain for quantification of HSP90α, which will make the quantification results standardized and comparable regardless of the quantitative methods.

Double-Labeling Method for Visualization and Quantification of Membrane-Associated Proteins in Lactococcus lactis.

Lactococcus lactis displaying recombinant proteins on its surface can be used as a potential drug delivery vector in prophylactic medication and therapeutic treatments for many diseases. These applications enable live-cell mucosal and oral administration, providing painless, needle-free solutions and triggering robust immune response at the site of pathogen entry. Immunization requires quantitative control of antigens and, ideally, a complete understanding of the bacterial processing mechanism applied to the target proteins. In this study, we propose a double-labeling method based on a conjugated dye specific for a recombinantly introduced polyhistidine tag (to visualize surface-exposed proteins) and a membrane-permeable dye specific for a tetra-cysteine tag (to visualize cytoplasmic proteins), combined with a method to block the labeling of surface-exposed tetra-cysteine tags, to clearly obtain location-specific signals of the two dyes. This allows simultaneous detection and quantification of targeted proteins on the cell surface and in the cytoplasm. Using this method, we were able to detect full-length peptide chains for the model proteins HtrA and BmpA in L. lactis, which are associated with the cell membrane by two different attachment modes, and thus confirm that membrane-associated proteins in L. lactis are secreted using the Sec-dependent post-translational pathway. We were able to quantitatively follow cytoplasmic protein production and accumulation and subsequent export and surface attachment, which provides a convenient tool for monitoring these processes for cell surface display applications.

Quantitation of reduced IL-33 levels in human serum: mitigating interference from endogenous binding partners.

Aim: IL-33 is a potential therapeutic target but commercially available assays for the quantitation of systemic IL-33 have poor reliability. Results: In commercial IL-33 kits, interference from endogenous binding partners (e.g., soluble ST2) causes under-quantitation. Mitigating this required acid dissociation and addition of the detection reagent simultaneously with the capture step. This enabled detection of total, reduced (active) levels of IL-33 in human serum (LLOQ 6.25 pg/ml). Conclusion: Acid treatment of serum samples dissociates IL-33 from endogenous binding partners, increasing soluble ST2 tolerance to >1000 ng/ml. The modified method was specific for reduced endogenous IL-33. Analysis of over 300 samples from individuals with and without asthma and with different smoking status revealed no difference in serum IL-33.

Ackr3-Venus knock-in mouse lights up brain vasculature.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits ßarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

nES-DMA with Charge-reduction based on Soft X-ray Radiation: Analysis of a Recombinant Monoclonal Antibody.

Due to the fast growing importance of monoclonal antibodies in biomedical research, bioanalytics and human therapy, sensitive, fast and reliable methods are needed to monitor their production, target their characteristics, and for their final quality control. Application of a nano electrospray (nES) with soft X-ray radiation (SXR) based charge reduction and differential mobility analysis (DMA, aka nano electrospray gas-phase electrophoretic mobility molecular analysis, nES GEMMA) allows the size-separation and detection of macromolecules and (bio-)nanoparticles from a few nm up to several hundreds of nm in diameter in a native-like environment. The current study focuses on the analysis of a 148 kDa recombinant monoclonal antibody (rmAb) with the above mentioned instrumental setup and applying an universal detector, i.e. a water-based condensation particle detector (CPC). Next to the intact rmAb, its aggregates and fragment products after digestion with IdeS protease were analyzed. Additionally, influence of temperature treatment and pH variation on the stability of the rmAb was monitored. In this context, changes in electrophoretic mobility diameter (EMD) values, peak shape, and signal intensity based on particle numbers were of interest. Molecular weights calculated by application of a correlation derived from respective standard protein compounds were compared to mass spectrometric values and were found to be in good accordance. To conclude, we demonstrate that nES-DMA is a valuable tool in the characterization and quality control of rmABs.

The COP9 signalosome subunit 3 is necessary for early embryo survival by way of a stable protein deposit in mouse oocytes.

Investigations of genes required in early mammalian development are complicated by protein deposits of maternal products, which continue to operate after the gene locus has been disrupted. This leads to delayed phenotypic manifestations and underestimation of the number of genes known to be needed during the embryonic phase of cellular totipotency. Here we expose a critical role of the gene Cops3 by showing that it protects genome integrity during the 2-cell stage of mouse development, in contrast to the previous functional assignment at postimplantation. This new role is mediated by a substantial deposit of protein (94th percentile of the proteome), divided between an exceptionally stable cortical rim, which is prevalent in oocytes, and an ancillary deposit in the embryonic nuclei. Since protein abundance and stability defeat prospects of DNA- or RNA-based gene inactivation in oocytes, we harnessed a classical method next to an emerging method for protein inactivation: antigen masking (for functional inhibition) versus TRIM21-mediated proteasomal degradation, also known as 'Trim away' (for physical removal). Both resulted in 2-cell embryo lethality, unlike the embryos receiving anti-green fluorescent protein. Comparisons between COPS3 protein-targeted and non-targeted embryos revealed large-scale transcriptome differences, which were most evident for genes associated with biological functions critical for RNA metabolism and for the preservation of genome integrity. The gene expression abnormalities associated with COPS3 inactivation were confirmed in situ by the occurrence of DNA endoreduplication and DNA strand breaks in 2-cell embryos. These results recruit Cops3 to the small family of genes that are necessary for early embryo survival. Overall, assigning genes with roles in embryogenesis may be less safe than assumed, if the protein products of these genes accumulate in oocytes: the inactivation of a gene at the protein level can expose an earlier phenotype than that identified by genetic techniques such as conventional gene silencing.

Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Analysis of Protein-Protein Interactions by Protein Microarrays.

The analysis of the proteome and the interactome would be useful for a better understanding of the pathophysiology of several disorders, allowing the identification of potential specific markers for early diagnosis and prognosis, as well as potential targets of intervention. Among different proteomic approaches, high-density protein microarrays have become an interesting tool for the screening of protein-protein interactions and the interactome definition of disease-associated dysregulated proteins. This information might contribute to the identification of altered signaling pathways and protein functions involved in the pathogenesis of a disease. Remarkably, protein microarrays have been already satisfactorily employed for the study of protein-protein interactions in cancer, allergy, or neurodegenerative diseases. Here, we describe the utilization of recombinant protein microarrays for the identification of protein-protein interactions to help in the definition of disease-specific dysregulated interactomes.

Simple, rapidly electroassembled thiolated PEG-based sensor interfaces enable rapid interrogation of antibody titer and glycosylation.

Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and enable near real-time assessment of antibody titer and N-linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal ß-galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer. Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T. sinensis.

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) is an arterivirus newly discovered in Chinese softshell turtles. Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles. In this study, a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system, and its polyclonal antibody was generated. The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay (dot-ELISA). The distribution of TSHSV in different organs of T. sinensis was examined by immunohistochemistry (IHC) and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed, and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles. The IHC assay indicated that the virus was highly localized in various cells, including intestinal lymphocytes, enterocytes, kidney epithelial cells, spleen cells, lung macrophages, and cardiomyocytes. The qRT-PCR analysis revealed that TSHSV was detected in all organs tested, including the lungs, liver, kidneys, spleen, and heart. The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group. The interferon-stimulated genes (ISGs), myxovirus resistance protein 2 (MX2) and radical S-adenosyl methionine domain containing 2 (RSAD2) were highly upregulated in all groups of infected turtles. Antibody-dependent enhancement (ADE) seemed to occur after stimulation by the polyclonal antibody, because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group. Overall, these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

Autophagy-inducing peptide increases CHO cell monoclonal antibody production in batch and fed-batch cultures.

The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy ("self-eating") is a cellular process that allows cells to mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy-inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed-batch serum-free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1-4 µM AIP enhanced productivity in a concentration-dependent manner. Cell-specific productivity increased up to 1.8-fold in batch cultures, while in fed-batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.

A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants.

A reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The method showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/mL, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.

Plant molecular farming for the production of valuable proteins - Critical evaluation of achievements and future challenges.

Recombinant proteins play an important role in many areas of our lives. For example, recombinant enzymes are used in the food and chemical industries and as high-quality proteins for research, diagnostic and therapeutic applications. The production of recombinant proteins is still dominated by expression systems based on microbes and mammalian cells, although the manufacturing of recombinant proteins in plants - known as molecular farming - has been promoted as an alternative, cost-efficient strategy for three decades. Several molecular farming products have reached the market, but the number of success stories has been limited by industrial inertia driven by perceptions of low productivity, the high cost of downstream processing, and regulatory hurdles that create barriers to translation. Here, we discuss the technical and economic factors required for the successful commercialization of molecular farming, and consider potential future directions to enable the broader application of production platforms based on plants.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

Respuesta inmunológica a varias proteínas del H. pylori en pacientes guatemaltecos / Inmune response to various H. pylori proteins in Guatemalan patients

Se determinó la respuesta inmunológica a proteínas recombinantes de Helicobacter pylori en pacientes dis-pépticos (adultos y niños), pacientes con cáncer gástrico y sus familiares asintomáticos adultos viviendo con ellos. Se utilizó la prueba recomLine® Helicobacter IgG e IgA, y con base en el reconocimiento de los factores de virulencia VacA y CagA se determinó si la cepa de H. pylori era de tipo I o II. El análisis de los datos fue descriptivo y analítico y se estimaron los intervalos de confianza de 95%, con un nivel de error de 0.05 y Odds ratio. El 58.7% (121/206) de los pacientes presentó la bacteria en tinción histológica de biopsia, positividad que disminuyó con la edad y daño histológico. La frecuencia de la respuesta a los anticuerpos IgG fue mayor que IgA, en ambos casos ésta fue menor en los niños. Las proteínas del H. pylori más reconocidas tanto por IgA como IgG fueron VacA y CagA, y la respuesta a las otras proteínas investigadas fue mayor al aumentar el daño histológi-co. La cepa tipo I fue la que predominó en la población en estudio con 66% (136/206). Se deben continuar con los estudios de prevalencia de la cepa tipo I del H. pylori y del reconocimiento de sus antígenos en la población guatemalteca a fin de determinar su utilidad en el diagnóstico y pronóstico de la infección.

The immune response to recombinant Helicobacter pylori proteins was determined in dyspeptic patients (adults and children), patients with gastric cancer and their asymptomatic adults' relatives living with them. The recomLine® Helicobacter IgG and IgA test was used and based on the recognition of the virulence factors VacA and CagA, it was determined whether the H. pylori strain was type I or II. The data analysis was descriptive and analytic, and 95% confidence intervals were estimated, with an error level of 0.05, and Odds ratio. The patients that presented the bacterium in histological biopsy were 58.7% (121/206), positivity that decreased with age and histological damage. The frecuency of response to IgG antibodies was higher than IgA, in both cases it was lower in children. VacA and CagA were the H. pylori proteins most recognized by both IgA and IgG and it was observed that the number of recognized proteins was greater with increasing histological damage. The type I strain was the one that predominated in the study population 66% (136/206). Prevalence studies of the type I strain of H. pylori ant the recognition of its antigens in the Guatemalan population should continue in order to determine its usefulness in the diagnosis and prognosis of infection.

Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 protein (CHIK-VP) as an example. The hydrophobic quantum dots (QDs) embedded in the lipid bilayer of liposome and methylene blue (MB) encapsulated in the inner core of liposomes played a role of dual-signaling modulator. After CHIK-VP addition, the nanocomposites and APTES-coated Fe3O4 nanoparticles (Fe3O4 NPs) were conjugated with antibodies to form a sandwich structure and separated from the medium magnetically. The nanoconjugates have been burst out by chloroform as surfactant, and both the QDs and MB are released from the liposome and were then monitored through changes in the fluorescence and electrochemical signals, respectively. These two fluorometric and electrochemical signals alteration quantified the CHIK-VP in the range of femtogram to nanogram per milliliter level with a LOD of 32 fg mL-1, making this liposomal system a potential matrix in a virus detection platform. Graphical abstract.

Synthesis, characterization and application of magnetoferritin nanoparticle by using human H chain ferritin expressed by Pichia pastoris.

Protein-based nanoparticles have developed rapidly in areas such as drug delivery, biomedical imaging and biocatalysis. Ferritin possesses unique properties that make it attractive as a potential platform for a variety of nanobiotechnological applications. Here we synthesized magnetoferritin (P-MHFn) nanoparticles for the first time by using the human H chain of ferritin that was expressed by Pichia pastoris (P-HFn). Western blot results showed that recombinant P-HFn was successfully expressed after methanol induction. Transmission electron microscopy (TEM) showed the spherical cage-like shape and monodispersion of P-HFn. The synthesized magnetoferritin (P-MHFn) retained the properties of magnetoferritin nanoparticles synthesized using HFn expressed by E. coli (E-MHFn): superparamagnetism under ambient conditions and peroxidase-like activity. It is stable under a wider range of pH values (from 5.0 to 11.0), likely due to post-translational modifications such as N-glycosylation on P-HFn. In vivo near-infrared fluorescence imaging experiments revealed that P-MHFn nanoparticles can accumulate in tumors, which suggests that P-MHFn could be used in tumor imaging and therapy. An acute toxicity study of P-MHFn in Sprague Dawley rats showed no abnormalities at a dose up to 20 mg Fe Kg-1 body weight. Therefore, this study shed light on the development of magnetoferritin nanoparticles using therapeutic HFn expressed by Pichia pastoris for biomedical applications.

Clinical implications of C6 complement component deficiency.

Background: Terminal complement component deficiencies are risk factors for neisserial infections. Objective: To review the clinical characteristics, the diagnosis and the management of patients with a terminal complement component deficiency. Methods: Pertinent articles were selected and reviewed in relation to a case presentation of C6 deficiency. Results: A case of a 56-year old patient with a history of meningitis, chronic rash, and C6 deficiency was presented, followed by discussion of clinical characteristics, diagnosis, and management of terminal complement component deficiencies. Clinical pearls and pitfalls were reviewed for the practicing allergist/immunologist and fellow-in-training. Conclusion: C6 deficiency is the most common terminal complement component deficiency and can present later in age with N. meningitidis infections. Patients can be screened for terminal complement component deficiency by checking CH50.

Acrolein in cigarette smoke attenuates the innate immune responses mediated by surfactant protein D.

BACKGROUND: Surfactant proteins (SP) A and D belong to collectin family proteins, which play important roles in innate immune response in the lung. We previously demonstrated that cigarette smoke (CS) increases the acrolein modification of SP-A, thereby impairing the innate immune abilities of this protein. In this study, we focused on the effects of CS and its component, acrolein, on the innate immunity role of another collectin, SP-D. METHODS: To determine whether aldehyde directly affects SP-D, we examined the lungs of mice exposed to CS for 1 week and detected aldehyde-modified SP-D using an aldehyde reactive probe. The structural changes in CS extract (CSE) or acrolein-exposed recombinant human (h)SP-D were determined by western blot, liquid chromatography-electrospray ionization tandem mass spectrometry, and blue native-polyacrylamide gel electrophoresis analyses. Innate immune functions of SP-D were determined by bacteria growth and macrophage phagocytosis. RESULTS: Aldehyde-modified SP-D as well as SP-A was detected in the lungs of mice exposed to CS for 1 week. Exposure of hSP-D to CSE or acrolein induced an increased higher-molecular -weight of hSP-D and acrolein induced modification of five lysine residues in hSP-D. These modifications led to disruption of the multimer structure of SP-D and attenuated its ability to inhibit bacterial growth and activate macrophage phagocytosis. CONCLUSION: CS induced acrolein modification in SP-D, which in turn induced structural and functional defects in SP-D. GENERAL SIGNIFICANCE: These results suggest that CS-induced structural and functional defects in SP-D contribute to the dysfunction of innate immune responses in the lung following CS exposure.

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells.

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.