Native Molecule Research Articles

Abstract 4841: Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer

Abstract Introduction: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colon cancer, but can cause serious gastrointestinal (GI), cardiovascular (CV), and renal side effects. Naproxen has a relatively favorable CV profile, but is notorious for its GI toxicity. To improve its safety profile, we developed NOSH-naproxen, where we linked nitric oxide (NO) and hydrogen sulfide (H2S) releasing moieties to the native molecule, the rational being the observations that NO and H2S may modulate some components of the local mucosal defense systems which should lead to reduced GI toxicity. Indeed, we recently reported that NOSH-naproxen displayed enhanced GI safety, with potent anti-inflammatory, analgesic, anti-pyretic, and anti-platelet properties. Here we describe the effects of NOSH-naproxen on the growth properties of several human colon cancer cell lines, and on its molecular targets in a xenograft mouse model of colon cancer. Methods: NOSH-naproxen was synthesized by us with 1H-NMR verification. Colon cancer cell lines: HT-29, HCT 15, and SW480; Cell growth: MTT. Xenografts: Male athymic nude (NU/NU) mice (N = 15) were implanted s.c. in the right flank with SW480 cells, when the tumors reached an average sizes of ∼100 mm3, the mice were randomly divided into 3 groups and gavaged daily with either vehicle (0.5% CMC) or equimolar concentrations of NOSH-naproxen (100 mg/kg) or naproxen (45 mg/kg). Tumor volume was measured every three days; after 30 days the mice were sacrificed, tumors collected, weighed, photographed and stored in formalin for IHC studies. Results: NOSH-naproxen inhibited the growth of HT-29, HCT 15, and SW480 cells with IC50 values of 90 ± 10, 100 ± 8, and 98 ± 7 nM at 24 hr, respectively. The corresponding IC50s for naproxen were 2800 ± 165, 2950 ± 215, and 3110 ± 185 nM. This represents an enhanced potency of ∼30,000-fold in the 3 cell lines at 24 h. In xenografts, all the mice treated with naproxen died within 2 weeks from what appeared to be GI bleeding. However, the NOSH-naproxen treated mice did not show any overt signs of toxicity. At sacrifice, NOSH-naproxen-treated mice had a mean reduction in tumor volume of 82%, and a mean reduction in tumor mass of 55%. NOSH-naproxen inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction of apoptosis (increased number of TUNEL positive cells). NF-êB, activated in untreated xenografts was significantly inhibited by NOSH-naproxen. Other molecular targets affected by NOSH-naproxen were: induction of iNOS, significant reduction in FoxM1, and increases in p53. Conclusions: NOSH-naproxen is more potent and efficacious than naproxen and appears to be significantly safer. It targets parameters important in determining cellular kinetics, inflammation, and signaling pathways important in the carcinogenic process. Citation Format: Pascale L. Duvalsaint, Mitali Chattopadhyay, Ravinder Kodela, Khosrow Kashfi. Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer. [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 4841.

Neurotrophic factor small-molecule mimetics mediated neuroregeneration and synaptic repair: emerging therapeutic modality for Alzheimer's disease.

Alzheimer’s disease (AD) is an incurable and debilitating chronic progressive neurodegenerative disorder which is the leading cause of dementia worldwide. AD is a heterogeneous and multifactorial disorder, histopathologically characterized by the presence of amyloid β (Aβ) plaques and neurofibrillary tangles composed of Aβ peptides and abnormally hyperphosphorylated tau protein, respectively. Independent of the various etiopathogenic mechanisms, neurodegeneration is a final common outcome of AD neuropathology. Synaptic loss is a better correlate of cognitive impairment in AD than Aβ or tau pathologies. Thus a highly promising therapeutic strategy for AD is to shift the balance from neurodegeneration to neuroregeneration and synaptic repair. Neurotrophic factors, by virtue of their neurogenic and neurotrophic activities, have potential for the treatment of AD. However, the clinical therapeutic usage of recombinant neurotrophic factors is limited because of the insurmountable hurdles of unfavorable pharmacokinetic properties, poor blood–brain barrier (BBB) permeability, and severe adverse effects. Neurotrophic factor small-molecule mimetics, in this context, represent a potential strategy to overcome these short comings, and have shown promise in preclinical studies. Neurotrophic factor small-molecule mimetics have been the focus of intense research in recent years for AD drug development. Here, we review the relevant literature regarding the therapeutic beneficial effect of neurotrophic factors in AD, and then discuss the recent status of research regarding the neurotrophic factor small-molecule mimetics as therapeutic candidates for AD. Lastly, we summarize the preclinical studies with a ciliary neurotrophic factor (CNTF) small-molecule peptide mimetic, Peptide 021 (P021). P021 is a neurogenic and neurotrophic compound which enhances dentate gyrus neurogenesis and memory processes via inhibiting leukemia inhibitory factor (LIF) signaling pathway and increasing brain-derived neurotrophic factor (BDNF) expression. It robustly inhibits tau abnormal hyperphosphorylation via increased BDNF mediated decrease in glycogen synthase kinase-3β (GSK-3β, major tau kinase) activity. P021 is a small molecular weight, BBB permeable compound with suitable pharmacokinetics for oral administration, and without adverse effects associated with native CNTF or BDNF molecule. P021 has shown beneficial therapeutic effect in several preclinical studies and has emerged as a highly promising compound for AD drug development.

Insights in understanding aggregate formation and dissociation in cation exchange chromatography for a structurally unstable Fc-fusion protein

Cation-exchange chromatography (CEX) of a structurally unstable Fc-fusion protein exhibited multi-peak elution profile upon a salt-step elution due to protein aggregation during intra-column buffer transition where low pH and high salt coexisted. The protein exhibited a single-peak elution behavior during a pH-step elution; nevertheless, the levels of soluble aggregates (i.e. high molecular weight species, HMW) in the CEX eluate were still found up to 12-fold higher than that for the load material. The amount of the aggregates formed upon the pH-step elution was dependent on column loading with maximum HMW achieved at intermediate loading levels, supporting the hypothesis that the aggregation was the result of both the conformational changes of the bound protein and the solution concentration of the aggregation-susceptible proteins during elution. Factors such as high load pH, short protein/resin contact time, hydrophilic resin surface, and weak ionizable ligand were effective, to some extent, to reduce aggregate formation by improving the structural integrity of the bound protein. An orthogonal technique, differential scanning fluorimetry (DSF) using Sypro Orange dye confirmed that the bound protein exposed more hydrophobic area than the native molecule in free solution, especially in the pH 4–5 range. The Sypro Orange dye study of resin surface property also demonstrated that the poly[styrene-divinylbenzene]-based Poros XS with polyhydroxyl surface coating is more hydrophobic compared to the agarose-based CM Sepharose FF and SP Sepharose FF. The hydrophobic property of Poros XS contributed to stronger interactions with the partially unfolded bound protein and consequently to the higher aggregate levels seen in Poros XS eluate. This work also investigates the aggregation reversibility in CEX eluate where up to 66% of the aggregates were observed to dissociate into native monomers over a period of 120h, and links the aggregate stability to such conditions as resin surface properties and charged ligand type. Experimental data was correlated semi-quantitatively with theoretical protein charge and hydrophobicity calculations using homology modeling within the BIOVIA Discovery Studio software. Finally, an arginine-sulphopropyl (Arg-SP) agarose resin immobilized with multi-functional ligands was prepared to verify the proposed hypothesis and to eliminate the aggregate formation. The findings of this work provide general insights in understanding aggregate formation and dissociation for structurally unstable proteins in the CEX step.

Chondrogenic Differentiation of Mesenchymal Stem Cells on Glycosaminoglycan-Mimetic Peptide Nanofibers.

Glycosaminoglycans (GAGs) are important extracellular matrix components of cartilage tissue and provide biological signals to stem cells and chondrocytes for development and functional regeneration of cartilage. Among their many functions, particularly sulfated glycosaminoglycans bind to growth factors and enhance their functionality through enabling growth factor-receptor interactions. Growth factor binding ability of the native sulfated glycosaminoglycans can be incorporated into the synthetic scaffold matrix through functionalization with specific chemical moieties. In this study, we used peptide amphiphile nanofibers functionalized with the chemical groups of native glycosaminoglycan molecules such as sulfonate, carboxylate and hydroxyl to induce the chondrogenic differentiation of rat mesenchymal stem cells (MSCs). The MSCs cultured on GAG-mimetic peptide nanofibers formed cartilage-like nodules and deposited cartilage-specific matrix components by day 7, suggesting that the GAG-mimetic peptide nanofibers effectively facilitated their commitment into the chondrogenic lineage. Interestingly, the chondrogenic differentiation degree was manipulated with the sulfonation degree of the nanofiber system. The GAG-mimetic peptide nanofibers network presented here serve as a tailorable bioactive and bioinductive platform for stem-cell-based cartilage regeneration studies.

Imaging mass spectrometry for the precise design of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a class of immunotherapeutic agents that enable the delivery of cytotoxic drugs to target malignant cells. Because various cancers and tumour vascular endothelia strongly express anti-human tissue factor (TF), we prepared ADCs consisting of a TF-specific monoclonal antibody (mAb) linked to the anticancer agent (ACA) monomethyl auristatin E (MMAE) via a valine-citrulline (Val-Cit) linker (human TF ADC). Identifying the most efficient drug design in advance is difficult because ADCs have complicated structures. The best method of assessing ADCs is to examine their selectivity and efficiency in releasing and distributing the ACA within tumour tissue. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) can be used to directly detect the distributions of native molecules within tumour tissues. Here, MALDI-IMS enabled the identification of the intratumour distribution of MMAE released from the ADC. In conclusion, MALDI-IMS is a useful tool to assess ADCs and facilitate the optimization of ADC design.

(Invited) Non-Covalent Functionalization of Single Wall Carbon Nanotubes with Engineered Proteins for Targeted Subcellular Delivery

Single wall carbon nanotubes (SWCNTs) can be non-covalently dispersed with surfactants, polymers and proteins. We have observed that protein coated SWCNTs enter numerous cell types by the millions, more than an order of magnitude more than polymer coated SWCNTs. In addition to increasing uptake, protein coatings can impart function to nanomaterials. While many contemporary studies have focused on using proteins to target SWCNTs and nanomaterials to specific cells, we utilize proteins to target SWCNTs to regions within cells. Here, we will show the utility of modified albumin proteins and engineered native cellular proteins as dispersing agents for SWCNTs. To localize SWCNTs to the nucleus we dispersed SWCNTs with the tail domain of a nucleoskeletal protein lamin B1 (LB1) engineered from the full-length LMNB1 cDNA. The low molecular weight globular protein has a central hydrophobic core for nanotube association and stabilization and an exposed nuclear localization sequence to promote active nuclear import. SWCNTs-LB1 enter HeLa cells and localize to the nucleus of cells; we visualize localization with Raman spectroscopy and NIR fluorescence imaging of SWCNTs and interaction with DNA by fluorescence lifetime imaging microscopy. We have used similar techniques to show cellular uptake of bovine serum albumin (BSA)-coated SWCNTs within cells. We see localization of SWCNTs-BSA within the endosomal and metabolic processing compartments. Similarly to LB1, BSA is a small globular protein, and the hydrophobic cleft of the BSA binds to SWCNTs. BSA has smaller hydrophobic regions, independent of the hydrophobic cleft, that can be loaded with drugs or fluorophores. In vitro, we show that denaturation or enzymatic processing of the BSA releases the small molecules. SWCNTs-BSA in which the BSA are pre-loaded with rhodamine drastically increases small molecule delivery in culture. We have determined spatial and concentration distribution of rhodamine within the cells, and signal is both coincident and distinct from SWCNTs as measured by NIR fluorescence and Raman. We demonstrate efficacy of this approach by delivering a fluorescent chemotherapeutic drug daunomycin that reduces proliferation in cancer cells. Together, our results demonstrates a pathway to increase the delivery of a wide variety of drugs to cells through SWCNTs coated with albumin pre-loaded with drug molecules. The complexity of protein structures allows for multimodal modification and manipulation of cellular processes in addition to SWCNT dispersion. The modification of native cellular proteins as non-covalent dispersing agents to provide specific transport opens new possibilities to utilize both SWCNT and protein properties for multifunctional subcellular targeting applications. Specifically, nuclear targeting will allow delivery of anticancer therapies, genetic treatments, or DNA, which in turn will promote development of novel cellular therapies. Using the transport properties of albumins and intracellular processing by enzymes allows delivery of native molecules and drugs.

Supporting Data for Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs.

Although nanoparticulate drug delivery systems (NDDSs) can preferentially accumulate in tumors, active targeting by targeting ligands (e.g. monoclonal antibody) is necessary for increasing its targeting efficacy in vivo. We conjugated mAb198.3 on the SiO2@AuNP system surface to make it obtain active targeting efficacy. The FAT1 targeting capability of SiO2@AuNP system is the first issue to be solved. Thus, flow cytometry analysis was attempted to demonstrate that the SiO2@AuNP system could bind to native FAT1 molecules on the surface of Colo205 cells. Also, together with the drug release behavior study of self-decomposable SiO2 NPs, the continuous morphological evolution needed to be clarified. Therefore, to characterize the morphological evolution in vitro, we analyzed the morphology of inner self-decomposable NPs in different time intervals using transmission electron microscopy (TEM). A more comprehensive analysis of this data may be obtained from the article “Multifunctional all-in-one drug delivery systems for tumor targeting and sequential release of three different anti-tumor drugs” in Biomaterials.

A preliminary study on the antibacterial mechanism of Tegillarca granosa hemoglobin by derived peptides and peroxidase activity.

The blood clam, Tegillarca granosa, is one of the few bivalve molluscs containing hemoglobin (Hb). In the present study, we purified two types of T.granosa hemoglobin, Tg-HbI and Tg-HbII, using size exclusion chromatography and measured their antibacterial and peroxidase activities. We also tested antibacterial activities of peptides prepared by trypsin digestion of purified Tg-Hb and reversed-phase high-performance liquid chromatography purification. Purified Tg-HbI and Tg-HbII showed antibacterial activity against Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Bacillus firmus, with differences in minimal inhibitory concentrations (MICs), but lacked antibacterial activity against Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Staphylococcus aureus. In contrast, 7 Tg-Hb derived peptides exhibited varying degrees of antibacterial activity against V.alginolyticus (MICs: 12-200μg/ml), V.parahaemolyticus (11-100μg/ml) and V.harveyi (1-200μg/ml). The antibacterial activity of Hb derived peptides was confirmed by fluorescence microscopy. In addition, peroxidase activity was detected in Tg-HbI and Tg-HbII. The results indicated that in addition to functioning as a respiratory protein T.granosa hemoglobins likely play a role in host antibacterial defense probably via a peroxidase activity of native molecules and some internal peptides released from the proteins.

Improvement of a liposomal formulation with a native molecule: calcitriol

Calcitriol and doxorubicin co-loaded liposomes created improved cytotoxicity on Namalwa cells compared to doxorubicin loaded liposomes or free drug treatments.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Amaranth peptides with antithrombotic activity released by simulated gastrointestinal digestion

Amaranth protein isolate was obtained and subjected to simulated gastrointestinal digestion to evaluate its potential antithrombotic activity. The protein isolate did not present fibrin clotting inhibition at the concentrations studied, whereas the hydrolysate (DH% = 51.1 ± 3.8%) exhibited inhibition of fibrin coagulation, showing a dose–response behaviour (IC50 = 0.23 ± 0.02 mg/mL), confirming that the enzymatic treatment was able to release bioactive peptides from amaranth proteins. A fraction with high antithrombotic activity was obtained from this hydrolysate, and resulted to be three times more potent than its original sample (IC50 = 0.07 ± 0.01 mg/mL). The absorption of this active fraction was studied with an in vitro peptides transport assay through intestinal epithelium and it was observed that some peptides are able to cross the Caco2-TC7 cell monolayer. Potentially bioactive peptides were found after sequencing them, and informatics tools allowed us to select and locate in their native molecules those peptides prone to inhibit thrombin activity.

Molecular Allergen-Specific IgE Assays as a Complement to Allergen Extract–Based Sensitization Assessment

Molecular allergen-based component-resolved diagnostic IgE antibody tests have emerged in the form of singleplex assays and multiplex arrays. They use both native and recombinant allergen molecules, sometimes in combination with each other, to supplement allergen extract-based IgE antibody analyses. The total number of available allergenic molecules has reached a diagnostically useful level; however, more molecules are needed to cover all the clinically important allergen specificities. Thus, for the foreseeable future, molecular allergen-specific IgE analyses will remain a supplement for initial allergen extract-based IgE antibody analyses in the diagnostic workup of the allergic patient. As a spin-off, it will enable manufacturers to improve the quality of extracts for in vitro testing. The 2 most exciting diagnostic developments linked to component-resolved diagnostic tests are the possibility to increase diagnostic sensitivity by the inclusion of allergens that are underrepresented in the current extracts and in vitro assays and to increase the diagnostic specificity by taking the information on allergen cross-reactivity into account. Particularly the latter application is still under development. This requires additional studies on the clinical relevance of serological cross-reactivity.

Rapid Phytotransformation of Benzotriazole Generates Synthetic Tryptophan and Auxin Analogs in Arabidopsis.

Benzotriazoles (BTs) are xenobiotic contaminants widely distributed in aquatic environments and of emerging concern due to their polarity, recalcitrance, and common use. During some water reclamation activities, such as stormwater bioretention or crop irrigation with recycled water, BTs come in contact with vegetation, presenting a potential exposure route to consumers. We discovered that BT in hydroponic systems was rapidly (approximately 1-log per day) assimilated by Arabidopsis plants and metabolized to novel BT metabolites structurally resembling tryptophan and auxin plant hormones; <1% remained as parent compound. Using LC-QTOF-MS untargeted metabolomics, we identified two major types of BT transformation products: glycosylation and incorporation into the tryptophan biosynthetic pathway. BT amino acid metabolites are structurally analogous to tryptophan and the storage forms of auxin plant hormones. Critical intermediates were synthesized (authenticated by (1)H/(13)C NMR) for product verification. In a multiple-exposure temporal mass balance, three major metabolites accounted for >60% of BT. Glycosylated BT was excreted by the plants into the hydroponic medium, a phenomenon not observed previously. The observed amino acid metabolites are likely formed when tryptophan biosynthetic enzymes substitute synthetic BT for native indolic molecules, generating potential phytohormone mimics. These results suggest that BT metabolism by plants could mask the presence of BT contamination in the environment. Furthermore, BT-derived metabolites are structurally related to plant auxin hormones and should be evaluated for undesirable biological effects.

Early renal graft function deterioration in recipients with preformed anti-MICA antibodies: partial contribution of complement-dependent cytotoxicity.

We previously reported that preformed anti-MHC class I-related chain A (MICA) antibodies increase the risk for renal graft rejection and enhance the deleterious effect of PRA(+) status early after transplantation. We studied 727 kidney recipients. Days to reach optimal serum creatinine level, estimated glomerular filtration rate (eGFR) at Month 3 and chronic kidney disease (CKD) stages were recorded. Anti-MICA specificities and C1q binding were tested by solid-phase assay. Complement-dependent cytotoxicity (CDC) and flow cytometry (FC) cross-matches with HeLa and PMA/CD28-T-blasts were performed. PRA(+)MICA(+) recipients exhibited longer time to reach optimal serum creatinine level after transplantation (P = 0.005) and had the lowest eGFR at Month 3 (P = 0.006). PRA(+)MICA(+) status independently increased the risk for CKDT stage 5 at Month 3 [hazard ratio (HR) 4.92, P = 0.030]. Pre-transplant anti-MICA antibodies were polyspecific and showed stronger reactions when coexisting with anti-HLA antibodies (mean standard fluorescent intensity 112 157 ± 44 426 in HLA(+)MICA(+) sera versus 49 680 ± 33 116 in HLA(-)MICA(+) sera, P = 0.0006). Anti-AYVE supereplet reactivity was significantly higher in HLA(+)MICA(+) versus HLA(-)MICA(+) patients (P < 0.001) and significantly superior than anti-CMGWS supereplet within HLA(+)MICA(+) patients (P = 0.001). Three of 13 anti-MICA(+) pre-transplant sera were positive for the C1q binding assay; one of them (serum 3) exclusively recognized AYVE supereplet with a strong reactivity against MICA*027 antigen (same as MICA*008). Anti-MICA antibodies in anti-HLA-absorbed serum 3 bound native MICA molecules in MICA*008(+) HeLa and PMA/CD28-T-blasts and mediated cell death by activating complement. Preformed anti-MICA antibodies may occasionally be cytotoxic by fixing and activating complement. This way they might contribute to worse early kidney graft function.

Catalase-only nanoparticles prepared by shear alone: Characteristics, activity and stability evaluation

Catalase is a promising therapeutic enzyme; however, it carries risks of inactivation and rapid degradation when it is used in practical bioprocess, such as delivery in vivo. To overcome the issue, we made catalase-only nanoparticles using shear stress alone at a moderate shear rate of 217s−1 in a coaxial cylinder flow cell. Properties of nanoparticles, including particle size, polydispersity index and zeta potential, were characterized. The conformational changes of pre- and post-sheared catalase were determined using spectroscopy techniques. The results indicated that the conformational changes of catalase and reduction in α-helical content caused by shear alone were less significant than that by desolvation method. Catalase-only nanoparticles prepared by single shear retained over 90% of its initial activity when compared with the native catalase. Catalase nanoparticles lost only 20% of the activity when stored in phosphate buffer solution for 72h at 4°C, whereas native catalase lost 53% under the same condition. Especially, the activity of nanogranulated catalase was decreased only slightly in the simulated intestinal fluid containing α-chymotrypsin during 4h incubation at 37°C, implying that the catalase nanoparticle was more resistant to the degradation of proteases than native catalase molecules. Overall, catalase-only nanoparticles offered a great potential to stabilize enzymes for various pharmaceutical applications.

Genetically engineered fusion of MAP-1 and factor H domains 1-5 generates a potent dual upstream inhibitor of both the lectin and alternative complement pathways.

Inhibition of the complement cascade has emerged as an option for treatment of a range of diseases. Mannose-binding lectin/ficolin/collectin-associated protein (MAP-1) is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway. The central regulator of the alternative pathway (AP) is complement factor H (FH). Our aim was to design a dual upstream inhibitor of both human lectin and APs by fusing MAP-1 with a part of FH. There were 2 different recombinant chimeric proteins comprising full-length human MAP-1 and the first 5 N-terminal domains of human FH designed. The FH domains were orientated either in the N- or C-terminal part of MAP-1. The complement inhibition potential in human serum was assessed. Both chimeric constructs displayed the characteristics of the native molecules and bound to the PRMs with an EC50 of ∼ 2 nM. However, when added to serum diluted 1:4 in a solid-phase functional assay, only the first 5 N-terminal domains of complement FH fused to the C-terminal part of full-length MAP-1 chimeric construct were able to combine inhibition of lectin and AP activation with an half maximal inhibitory concentration of ∼ 100 and 20 nM, respectively. No effect was seen on the classical pathway. Fusion of MAP-1 with FH domains represents a novel therapeutic approach for selective targeting upstream and central complement activation at sites of inflammation.

Molecular Allergy Diagnostics: Analytical Features That Support Clinical Decisions.

Application of purified native and recombinant allergenic molecules into IgE antibody assays can improve analytical sensitivity and specificity for selected allergen specificities. They enhance analytical sensitivity by allowing assays to detect IgE antibodies with a lower limit of quantification (LoQ) to missing or poorly represented allergens in diagnostic extracts that are commonly used in vivo and in vitro. Use of selected allergenic molecules can help improve the clinician's prediction of the risk of a serious allergic reaction to stable allergens. They can provide diagnostic information to determine if a provocation challenge (e.g., oral food challenge) is indeed mandatory or not necessarily needed to support the final diagnostic decision. Suspected cross-reactivity based on the clinical history can be adjudicated by analyzing IgE antibodies to allergenic molecules from cross-reactive protein families. Finally, genuine primary sensitization can be identified by IgE antibody responses that are measured to selected allergenic molecules which are present in only one particular allergen source. After allergen-specific IgE detection, careful interpretation is required by the physician who knows the patient's history. Applying single allergen molecules, positive IgE antibody results are still only relevant in the case of corresponding objective symptoms. Subsequently, clinical relevance of such an IgE antibody test result must be determined by the clinician and not by the test itself. Because of their comprehensive nature, allergen extracts will remain the principal allergen source for diagnostic in vivo and in vitro assays of IgE antibody for many years. Judicious use of individual allergenic molecules in serum IgE assays may provide their most cost effective and efficient application for establishing a definitive diagnosis of human allergic disease.

Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

How amide hydrogens exchange in native proteins

Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.

Enzymatic synthesis, purification and in vitro antioxidant capacity of polyphenolic oxidation products from apple juice

5-O-Caffeoylquinic acid (CQA) and (−)-epicatechin (EC) commonly present in apple were oxidized with a polyphenoloxidase extract (PPO) from apple to produce oxidation products formed in the course of apple processing into juice. The oxidation products were characterized by RP-HPLC-ESI-MS. CQA homodimers and EC–CQA heterodimers were the main compounds produced as shown on the extracted ion LC-MS chromatograms of the corresponding deprotonated molecules at m/z 705 and 641, respectively. Thirteen fractions purified by semi preparative HPLC were obtained as pure compounds according to the MS spectrum. In vitro DPPH˙ radical scavenging activity of those purified oxidation products was evaluated and compared to the native parent molecules and Trolox. The results were discussed on the basis of structure–activity relationship. On a molar basis, heterodimers exhibited higher antioxidant potential than homodimers. However, they showed a lower activity than an equimolar mixture of the parent molecules. These results provide new information about antioxidant activities of polyphenolic oxidation products which have an impact on the nutritional value of apple juices and other apple-derived foods.