Dimethyl Suberimidate Research Articles

Self-directed molecular diagnostics (SdMDx) system for COVID-19 via one-pot processing

Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)− 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.

Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

Since its discovery in circulating blood seven decades ago, cell-free DNA (cfDNA) has become a highly focused subject in cancer management using liquid biopsy. Despite its clinical utility, the extraction of cfDNA from blood has many technical difficulties, including a low efficiency of recovery and long processing times. We introduced a magnetic bead-based cfDNA extraction method using homobifunctional crosslinkers, including dimethyl suberimidate dihydrochloride (DMS). Owing to its bifunctional nature, DMS can bind to DNA through either covalent or electrostatic bonding. By adopting amine-conjugated magnetic beads, DMS–DNA complexes can be rapidly isolated from blood plasma. Using standard washing and eluting processes, we successfully extracted cfDNA from plasma within 10 min. This method yielded a 56% higher extraction efficiency than that of a commercial product (QIAamp kit). Furthermore, the instant binding mechanism of amine coupling between the microbeads and DMS–DNA complexes significantly reduced the processing time. These results highlight the potential of this magnetic bead-based homobifunctional crosslinker platform for extraction of cfDNA from blood plasma.

Exploring the effects of the crosslink density on the physicochemical properties of collagen-based scaffolds

The optimization of collagen-based scaffolds for tissue engineering goes through the careful selection of the crosslinking method(s), which should impart the prerequisite mechanical and degradation properties without impairing the cell/tissue response. Here, we investigated the chemically effective (ρxch) and the elastically effective (ρxel) crosslink density of collagen-based scaffolds, induced by various crosslinking methods. The aim was to get a deeper insight into the influence of intramolecular and intermolecular crosslinks on several scaffold properties. Freeze-dried collagen matrices were crosslinked via a dehydrothermal treatment (DHT), and then treated with different chemical agents, including carbodiimide (EDC), glutaraldehyde (GTA), formaldehyde (FA), genipin (GP) and dimethyl suberimidate (DMS). Quantification of primary amines and stress-relaxation compressive tests were performed to evaluate ρxch and ρxel, respectively. Scaffolds were then assessed for their water uptake, thermal stability and in vitro resistance to enzymatic degradation. Interestingly, for the various crosslinking treatments ρxch was found to increase in the order DHT < DHT + GP < DHT + DMS < DHT + GTA < DHT + FA < DHT + EDC, while ρxel increased according to this slightly different trend: DHT < DHT + GP < DHT + DMS < DHT + EDC < DHT + GTA < DHT + FA. Indeed, treatment DHT + EDC induced a higher ρxch but a lower ρxel than aldehyde-based ones. This finding, together with the higher denaturation temperature (Td) of EDC-treated samples compared to others, suggested that zero-length EDC crosslinking promoted intramolecular crosslinks, along with intermolecular ones. Accordingly, the increase of Td was correlated with the increase of ρxch rather than ρxel, whereas the decrease in water uptake was consistent with the increase of ρxel, as expected. An exponential relationship between ρxel and the in vitro half-life was also determined.

Large Instrument- and Detergent-Free Assay for Ultrasensitive Nucleic Acids Isolation via Binary Nanomaterial.

Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.

Improved Reversible Cross-Linking-Based Solid-Phase RNA Extraction for Pathogen Diagnostics.

In this study, we developed an amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted (ADD) system as a novel binding strategy to improve the solid-phase extraction method for rapid and simple purification of RNA from biological samples including human cells and pathogenic bacteria. This ADD system is based on reversible cross-linking reactions between RNA and the silica matrix. The formation of robust covalent bonds protects RNA from both the sufferance of washing steps and isolation with ribonuclease (RNase)-rich samples, leading to the extraction of higher quality RNA. This improved RNA extraction system integrated with quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is evaluated for pathogen diagnostics. Compared to standard solid-phase extraction based commercial kits, this improved method shows highly enhanced sensitivity with 1000-fold higher sensitivity for human cells and 100-fold higher sensitivity for Brucella bacteria, according to the cycle threshold value of RT-qPCR. We envision that the ADD system can be tailored for commercial applications for RNA expression analysis in forensics studies, as well as for disease diagnostics in clinical applications.

Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes.

Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.

Rapid virus diagnostic system using bio-optical sensor and microfluidic sample processing

Respiratory infection diseases caused by viruses threaten public health worldwide. Although many detection techniques have been developed for virus analysis, they still lack sensitivity, simplicity, and rapidity and require separate sample preparation steps. In this study, we report a virus diagnostic system based on a bio-optical sensor combined with microfluidic sample processing for the rapid and sensitive detection of human adenovirus (HAdV). This system was assembled with a microfluidic-based viral DNA extraction technique and a silicon-based bio-optical sensor for systemically virus extraction and detection. First, viral DNA was obtained from HAdV samples using a novel DNA extraction technique, which is based on the integration of a novel non-chaotropic reagent (dimethyl suberimidate) with a disposable thin-film platform in a single microchannel. This platform can extract viral DNA from clinical samples within 30min and without any instruments. Second, the bio-optical sensor can detect up to 10 copies of HAdV in clinical samples within 30min. Finally, we validated the clinical utility of the virus diagnostic system in 13 human samples, including 10 samples with HAdV and three samples with another pathogen. This virus diagnostic system offers a rapid (<1h) and sensitive (100 times higher than qPCR) diagnostic platform for viral DNA analysis with low cost, simplicity, short assay time, and without the need for complex instruments. Therefore, this system enables the fast diagnosis of viral infection to control the spread of the virus and provide appropriate treatment in the field of respiratory infection diseases.

A single-tube approach for in vitro diagnostics using diatomaceous earth and optical sensor

Versatile, simple and efficient sample preparation is desirable for point-of-care testing of emerging diseases such as zoonoses, but current sample preparation assays are insensitive, labour-intensive and time-consuming and require multiple instruments. We developed a single-tube sample preparation approach involving direct pathogen enrichment and extraction from human specimens using diatomaceous earth (DE). Amine-modified DE was used to directly enrich a zoonotic pathogen, Brucella, in a large sample volume. Next, a complex of amine-modified DE and dimethyl suberimidate was used for nucleic acid extraction from the enriched pathogen. Using our single-tube approach, the pathogen can be enriched and extracted within 60min at a level of 1 colony formation unit (CFU) from a 1ml sample volume in the same tube. The performance of this approach is 10–100 times better than that of a commercial kit (102 to 103CFU/ml) but does not require a large centrifuge. Finally, we combined the single-tube approach with a bio-optical sensor for rapid and accurate zoonotic pathogen detection in human urine samples. Using the combination system, Brucella in human urine can be efficiently enriched (~ 8-fold) and the detection limit is enhanced by up to 100 times (1CFU/ml bacteria in urine) compared with the commercial kit. This combined system is fast and highly sensitive and thus represents a promising approach for disease diagnosis in the clinical setting.

Examining Properties of the C-Terminal Domain of Human Apolipoprotein A-I Utilizing a Chimeric Apolipoprotein

Human apolipoprotein A-I (apoA-I) is a 28 kDa exchangeable apolipoprotein that constitutes the primary protein of high density lipoprotein and plays a vital role in reverse cholesterol transport. ApoA-I comprises two structural domains: an α-helical N-terminal domain (NT, residues 1-189) and a less structured C-terminal domain (CT, residues 190-243). To evaluate the properties of the CT domain of apoA-I independently from the NT domain, a chimeric apolipoprotein was engineered in which residues 179-243 of apoA-I were attached to apolipophorin III (apoLp-III). ApoLp-III is a well-characterized insect apolipoprotein with a one domain structure composed of a bundle of α-helices. Two cysteine residues were introduced in apoLp-III to control opening of the helix bundle. ApoLp-III is monomeric but cross-linking studies with dimethyl suberimidate showed that the chimera self-associates similar to apoA-I. Far UV circular dichroism showed that the chimera displayed less α-helical content compared to apoLp-III, which is consistent with the addition of a less structured domain. Denaturation analysis using guanidine HCl showed that the stability of the chimera increased compared to apoLp-III. Functional analysis showed that the chimera was able to solubilize phospholipid vesicles at a faster rate than apoLp-III and that the percent calcein released from large unilamellar vesicles made of phosphatidylglycerol was substantially higher. Oxidized chimera protected LDL from phospholipase C-induced aggregation to a similar extent as apoLp-III but was far superior in its reduced state. Since the attachment of the CT domain of apoA-I to apoLp-III resulted in oligomerization and enhanced lipid binding, this demonstrated that these properties reside in the CT domain of apoA-I, and were transferable to an unrelated apolipoprotein. This demonstrates the potential to use the chimera as a tool to study the CT domain of apoA-I.

Small-angle X-ray scattering method to characterize molecular interactions: Proof of concept.

Characterizing biomolecular interactions is crucial to the understanding of biological processes. Existing characterization methods have low spatial resolution, poor specificity, and some lack the capability for deep tissue imaging. We describe a novel technique that relies on small-angle X-ray scattering signatures from high-contrast molecular probes that correlate with the presence of biomolecular interactions. We describe a proof-of-concept study that uses a model system consisting of mixtures of monomer solutions of gold nanoparticles (GNPs) as the non-interacting species and solutions of GNP dimers linked with an organic molecule (dimethyl suberimidate) as the interacting species. We report estimates of the interaction fraction obtained with the proposed small-angle X-ray scattering characterization method exhibiting strong correlation with the known relative concentration of interacting and non-interacting species.

Abstract 4571: Antitumor effects of an antibody (cetuximab)-targeted nanoparticle containing siRNA against EGFR

Abstract Objective: Effective therapies for lung cancer that have mutated EGFR remain elusive. Because of the success of antibody-drug-conjugates, we are exploring the use of antibody targeted nanoparticles (NP) containing siRNA to produce multifunctional therapeutics. Here, we investigate the use of cetuximab (CTX) (commercial antibody to EGFR) targeted NPs containing siRNA against EGFR (sequence is for a region of the mRNA conserved in all types of mutations) in H1975 NSCLC xenografts. Methods: We synthesized a cationic mucic acid polymer by polymerizing a derivative of mucic acid, mucic acid ethylenediamine, with dimethyl suberimidate. This polycation can self-assemble with siRNA to form the NP. A methoxy polyethylene glycol (PEG) was modified to have a 5-nitrophenyl boronic acid (5-nPBA) moiety at one end to attach to diol groups on mucic acid and stabilize the NP. 5-nPBA-PEG with a carboxylic acid end group was conjugated to CTX to target the NP to EGFR receptors on the surface of cancer cells. These polymers were mixed with siRNA to form NPs, characterized by dynamic light scattering and cryo-transmission electron microscopy for size, and zeta potential measurements for surface charge. The NP was tested in vivo against tumors grown with H1975 NSCLC cells that contain the L858R and T790M mutations. NPs dosed iv (tail vein) at 9.3 mg/kg CTX targeting agent and 5 mg/kg siEGFR were injected twice a week for two weeks into nude mice with H1975 NSCLC xenografts and the anti-tumor effects compared to 9.3 mg/kg CTX (dosed iv tail vein) alone. Results: The CTX-targeted cMAP-siRNA NPs are ca. 40 nm in diameter as determined by both dynamic light scattering and cryo-TEM. The NPs' surface charge is slightly negative at -3.5 mV in 10 mM phosphate buffered at pH 7.4 (surrogate for extracellular conditions in vivo). When the NPs are in 1 mM KCl at pH 5.5 (surrogate for intracellular, endocytosis localization), the NP zeta potential is +1 mV indicating detachment of 5-nPBA-PEG from the NP at acidic pH. Thus, the NP is stabilized and targeted until it is endocytosed, where it disassembles to release the siRNA payload. We confirmed delivery of siEGFR to the tumor following two NP doses via a real-time PCR assay. In contrast to CTX treated mice that showed tumor growth delay but ultimately had tumors that grew to over 1000 mm3, NP treated mice had tumors that remained below 200 mm3 53 days following completion of 4 doses. Conclusions: NPs containing full antibodies as targeting agents and siRNA payloads can be formulated into well defined, stable experimental therapeutics. The NP system used here has a pH-tunable 5-nPBA that allows for targeting and stabilizing the NP at a physiologic pH of 7.4. The targeting agent and PEG coating is able to detach from the NP at acidic pH like those found in endosomes, enabling the siRNA payload to escape and reach its site of action within the cell. These NPs produce significant tumor regression in vivo that is superior to CTX alone. Citation Format: Dorothy W. Pan, Mark E. Davis. Antitumor effects of an antibody (cetuximab)-targeted nanoparticle containing siRNA against EGFR. [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 4571. doi:10.1158/1538-7445.AM2014-4571

Polymeric hydrogel thin film synthesis via diffusion through a porous membrane

We report here a simple, versatile bi-cell-based platform for preparing crosslinked polymeric hydrogel thin films. Briefly, a nanoporous membrane is used to separate two solutions: one containing crosslinking molecules and the other containing oligomers. The crosslinking molecule diffuses through the membrane and reacts with the oligomer to form a polymeric film at the surface of the nanopore membrane. In this paper, a proof-of-concept experiment is described using crosslinkers with imidoester moieties (dimethyl 3,3′-dithiobispropionimidate (DTBP) or dimethyl suberimidate (DMS)) and chitosan oligomers. Crosslinking with DTBP or DMS also conferred degradability to the chitosan film. The film formation was confirmed and its morphology examined with electron microscopy. The chitosan film degradation was quantified by monitoring the transport of gold nanoparticles through the chitosan film after a degradation treatment. The film synthesis method presented here can potentially be used to prepare functional hydrogel thin films for biosensors, coatings and drug delivery systems in an inexpensive, high-throughput manner.

Imidate-based cross-linkers for structural proteomics: increased charge of protein and peptide ions and CID and ECD fragmentation studies.

Chemical cross-linking is an attractive low-resolution technique for structural studies of protein complexes. Distance constraints obtained from cross-linked peptides identified by mass spectrometry (MS) are used to construct and validate protein models. Amidinating cross-linkers such as diethyl suberthioimidate (DEST) have been used successfully in chemical cross-linking experiments. In this work, the application of a commercial diimidate cross-linking reagent, dimethyl suberimidate (DMS), was evaluated with model peptides and proteins. The peptides were designed with acetylated N-termini followed by random sequences containing two Lys residues separated by an Arg residue. After cross-linking reactions, intra- and intermolecular cross-linked species were submitted to CID and ECD dissociations to study their fragmentation features in the gas phase. Fragmentation of intramolecular peptides by collision induced dissociation (CID) demonstrates a unique two-step fragmentation pathway involving formation of a ketimine as intermediate. Electron capture and electron transfer dissociation (ECD and ETD) experiments demonstrated that the cyclic moiety is not dissociated. Intermolecular species demonstrated previously described fragmentation behavior in both CID and ECD experiments. The charge state distributions (CSD) obtained after reaction with DMS were compared with those obtained with disuccinimidyl suberate (DSS). CSDs for peptides and proteins were increased after their reaction with DMS, owing to the higher basicity of DMS modified species. These features were also observed in LC-MS experiments with bovine carbonic anhydrase II (BCA) after cross-linking with DMS and tryptic proteolysis. Cross-linked peptides derived from this protein were identified at high confidence and those species were in agreement with the crystal structure of BCA.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Hydrophobic and membrane permeable polyethylenimine nanoparticles efficiently deliver nucleic acids in vitro and in vivo.

Conjugation through primary amines is one of the most commonly used methods to modify cationic vectors for efficient gene delivery. Here, dimethyl suberimidate, a commercially available homobifunctional reagent bearing imidoesters at the termini, has been used to crosslink branched polyethylenimine (bPEI) into its nanoparticles (crosslinked PEI nanoparticles, CLP NPs) specifically through primary amines without altering the total charge on the resulting NPs for interaction with biomolecules and cell membranes. By varying the degree of crosslinking, a small series of CLP NPs was prepared and evaluated for their capability to deliver nucleic acids in vitro and in vivo. Physico-chemical characterization revealed the size of the NPs in the range of ∼152 to 210 nm with zeta potential ∼+35 to +38 mV. The plasmid DNA binding ability of these nanoparticles was examined by mobility shift assay, where the pDNA migration was found to be completely retarded by these NPs at an N/P ratio of 4 (cf. bPEI at N/P 3). In various mammalian cells, CLP/pDNA nanoplexes were not only found to be non-toxic but also exhibited significantly enhanced gene expression with one of the formulations, the CLP3/pDNA nanoplex, displaying the highest transfection efficiency, outperforming native bPEI and the selected commercial transfection reagents both in the presence and absence of serum. Further, the versatility of the vector, CLP3, was demonstrated by sequential delivery of GFP-specific siRNA to HEK293 cells, which resulted in ∼79% suppression of the target gene. Intracellular localization studies showed a significant population of the dual labeled nanoplex (CLP3/pDNA) in the nucleus in just 60 min of incubation. Luciferase reporter gene analysis in Balb/c mice post-intravenous administration of the CLP3/pDNA nanoplex showed the highest gene expression in their spleen. The study suggests that CLP NPs could be used as efficient gene delivery vectors for future gene therapy applications.

HMGB1-Facilitated p53 DNA Binding Occurs via HMG-Box/p53 Transactivation Domain Interaction, Regulated by the Acidic Tail

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic.

Effects of Autologous, Cross-Linked Erythrocytes on Isolated Hypoperfused Rabbit Heart Dynamics

The present study was aimed at assessing the effects of either red blood cells (RBC) or RBC cross-linked with the bifunctional dimethyl suberimidate reagent (C-RBC) on contractile force (CFo), heart rate (HR) and coronary flow (CF) of the isolated rabbit heart hypoperfused with RBC suspensions under 30 mm Hg constant pressure. RBC or C-RBC caused a rapid and marked reduction of CF, CFo and HR. In RBC-treated hearts, however, reperfusion with Tyrode solution partially restored the initial myocardial parameters, while in C-RBC-treated hearts a rapid impairment of diastolic relaxation with a subsequent, steady and increasing heart contracture was observed. Histological analysis showed that in C-RBC-perfused hearts either capillaries or precapillary arterioles were occluded by C-RBC in spite of extensive washings with Tyrode solution. These findings indicate that C-RBC impair coronary circulation markedly and irreversibly.

Inhibition of RNase L and RNA-dependent Protein Kinase (PKR) by Sunitinib Impairs Antiviral Innate Immunity

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC(50) values of 1.4 and 0.3 μM, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-β levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.

Purification of enzymatically inactive peptidylarginine deiminase type 6 from mouse ovary that reveals hexameric structure different from other dimeric isoforms

The murine peptidylarginine deiminase (PAD) has five isoforms encoded by different genes and participates in a variety of cellular functions through the citrullination of target proteins. The crystal structure of human PAD4 with a dimeric form was previously solved because of the enzyme’s relevance to rheumatoid arthritis. PAD6, abundant in mouse oocytes and eggs, is believed to take part in early events of embryogenesis, but its biochemical properties are little understood. Here we have purified and characterized a recombinant PAD6. A PAD6 cDNA was cloned from mouse ovary RNA and expressed in Escherichia coli through pET29 and pGEX vectors. When benzoyl-L-arginine ethyl ester was used as a substrate, no appreciable activity was detected with a cell homogenate under conditions where a human PAD4 cDNA caused significant activity. Both proteins were affinity-purified to near homogeneity. The circular dichroism spectra of PAD6 and human PAD4 were similar in the far ultraviolet region. On molecular sieving, PAD6 was eluted faster than human PAD4. The cross-linking of PAD6 with dimethyl suberimidate clearly showed six bands on an sodium dodecyl sulfate-polyacrylamide gel. These results indicate that PAD6 can constitute a hexameric structure. The purified PAD6 still showed no enzymatic activity. This unique structure and loss in enzymatic activity is strongly suggested to favor the formation of egg cytoplasmic sheets as the architectural protein.

Immobilization of Cell Wall Invertase Modified with Glutaraldehyde for Continuous Production of Invert Sugar

Yeast cell wall invertase (CWI) was modified with dimethyl suberimidate, glutaraldehyde, formaldehyde, and sodium periodate. Retained activity after modification was 45% for CWI modified with formaldehyde, 77% for CWI modified with sodium periodate, 80% for CWI modified with glutaraldehyde, and 115% for CWI modified with dimethyl suberimidate. Chemically modified and native CWIs showed significantly broad pH stability (pH 3-11), whereas after incubations at 50, 60, and 70 °C, CWI modified with glutaraldehyde showed the highest thermostability. Optimum pH for CWI modified with glutaraldehyde was between 4 and 5, whereas optimum temperature was at 60 °C. Comparison to CWI modified with glutaraldehyde after immobilization within alginate beads showed broader pH optimum (4.0-5.5) as well as broader temperature optimum (55-70 °C). Column bed reactor packed with the immobilized CWI modified with glutaraldehyde was successfully used for the 95% inversion of 60% (w/w) sucrose at the flow rate of 3 bed volumes per hour, pH 4.9, and 45 °C. A 1 month productivity of 3844 kg of inverted sugar/kg of the immobilisate was obtained.