Normal tissue injury from accidental or therapeutic exposure to high-dose radiation can cause severe acute and delayed toxicities, which result in mortality and chronic morbidity. Exposure to single ...high-dose radiation leads to a multi-organ failure, known as acute radiation syndrome, which is caused by radiation-induced oxidative stress and DNA damage to tissue stem cells. The radiation exposure results in acute cell loss, cell cycle arrest, senescence, and early damage to bone marrow and intestine with high mortality from sepsis. There is an urgent need for developing medical countermeasures against radiation injury for normal tissue toxicity. In this review, we discuss the potential of applying secretory extracellular vesicles derived from mesenchymal stromal/stem cells, endothelial cells, and macrophages for promoting repair and regeneration of organs after radiation injury.
The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate ...the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8
T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8
T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8
T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8
T-cell populations in vivo, which may serve prognostic and diagnostic roles.
Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional ...initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects.
Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.
The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with ...function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity--14% or less--between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology/threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton‐coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor ...for intestinal folate uptake. It is also involved in folate transport across the blood–brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo‐oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi‐functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system 3Hfolic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitonin‐solubilized non‐denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5‐HT3A receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma‐membrane PCFT functions as a monomeric protein.
Human proton‐coupled folate transporter (PCFT) was heterologously expressed in CHO cells and X. laevis oocytes. In both expression systems PCFT was functional and the predominant PCFT protomer on the plasma membrane was determined to be monomeric by both blue‐native PAGE and cross‐linking experiments.
Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, ...we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3′-125I-iodo-4′-azidopropiophenone (SADU-3-72). Based on inhibition of 125ISADU-3-72 binding, SADU-3-72 binds with high affinity (IC50 = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC50 = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with 125ISADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of 125ISADU-3-72 photoincorporation within nAChR subunit fragments containing M1–M2–M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1–M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu265, βLeu257) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr213 in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.
TFF2 is a small, secreted protein with anti-inflammatory properties. We previously have shown that TFF2 gene delivery via adenovirus (Ad-Tff2) suppresses colon tumor growth in colitis associated ...cancer. Therefore, systemic administration of TFF2 peptide could potentially provide a similar benefit. Because TFF2 shows a poor pharmacokinetic, we sought to modify the TFF2 peptide in a manner that would lower its clearance rate but retain bioactivity. Given the absence of a sequence-based prediction of TFF2 functionality, we chose to genetically fuse the C-terminus of TFF2 with the carboxyl-terminal peptide of human chorionic gonadotropin β subunit, and inserted into adenoviral vector that expresses Flag. The resulting Ad-Tff2-CTP-Flag construct translates into a TFF2 fused with two CTP and three Flag motifs. Administered Ad-Tff2-CTP-Flag decreased tumorigenesis and suppressed the expansion of myeloid cells in vivo. The fusion peptide TFF2-CTP-Flag delivered by adenovirus Ad-Tff2-CTP-Flag as well purified recombinant fusion TFF2-CTP-Flag was retained in the blood longer compared with wild-type TFF2 delivered by Ad-Tff2 or recombinant TFF2. Consistently, purified recombinant fusion TFF2-CTP-Flag suppressed expansion of myeloid cells by down-regulating cyclin D1 mRNA in vitro. Here, we demonstrate for the very first time the retained bioactivity and possible pharmacokinetic advantages of TFF2 with a modified C-terminus.
BackgroundImmunotherapies are highly promising and effective strategies for the treatment of cancer; however, continuing challenges persist, including 1) untargeted global immune modulation, ...resulting in serious side effects; 2) lack of therapeutics capable of in vivo expansion of tumor-specific T cells; 3) inability to visualize in vivo tumor-specific T cell responses; and 4) lack of flexible platforms to rapidly and efficiently explore new therapeutic strategies and immune-escape mechanisms. To address these challenges, we developed a novel class of precision biologics to treat cancer, autoimmune diseases and infectious diseases. We describe a modular platform constructed around an Fc-based covalent pMHC dimer, referred to as synTac (artificial synapse for T cell activation; also termed Immuno-STATs for Selective Targeting and Alteration of T cells), which selectively delivers different cargoes, including costimulatory, coinhibitory or cytokine signals and other modalities to primary T cells of defined specificity. The inherent modularity supports broad applications. Changing the encoded peptide enables targeting of different T cell specificities to address different diseases, while altering the cargo allows for evaluation of different co-modulatory mechanisms or the delivery of mechanistically informative probes.MethodsSortase A-mediated enzymatic coupling supported site-specific and stoichiometric installation of positron emission tomography (PET)-active radiolabels on synTacs to visualize the in vivo localization of antigen-specific T cells. The NSG humanized mouse model allowed for the evaluation of synTacs/Immuno-STATs to drive the in vivo antigen-specific expansion of human CD8 T cells.ResultsUsing radiolabeled synTacs/Immuno-STATs loaded with the appropriate peptides, we employed positron emission tomography to localize human papillomavirus (HPV16)-specific CD8 T cells to implanted HPV16-positive tumors in mice, as well as influenza A virus (IAV)-specific CD8 T cells in the lungs of IAV-infected mice. In vivo administration of HIV- and CMV-specific synTacs/Immuno-STATs to immunodeficient mice intrasplenically engrafted with human donor PBMCs resulted in the marked and selective expansion of HIV-specific and CMV-specific human CD8 T cells populating their spleens, respectively.ConclusionsWe demonstrate the remarkable flexibility of the synTacs/Immuno-STAT platform for addressing a broad range of applications, including the first report of the in vivo imaging of antigen-specific CD8 T cell populations and in vivo antigen-selective expansion of human CD8 T cells. These results suggest that, in addition to broad therapeutic applications, synTac/Immuno-STATs may provide prognostic/diagnostic information. Most notably, these results demonstrate the presence of synTacs/Immuno-STAT biologics in the tumor or infected tissues where they can elicit T cell restimulation and expansion necessary for target killing and enhanced therapeutic efficacy.
Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify ...determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with 3Hepibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, 3Hepibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.
A HEK-293 cell line that stably expresses mouse 5-HT3ARs containing a C-terminal extension that confers high-affinity binding of α-bungarotoxin (αBgTx) was established (αBgTx-5-HT3ARs) and used to ...purify αBgTx-5-HT3ARs in a lipid environment for use in structural studies using photoaffinity labeling. αBgTx-5-HT3ARs were expressed robustly (60 pmol of 3HBRL-43694 binding sites (∼3 μg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC50 = 5.3 ± 0.04 μM). While 125IαBgTx bound to the αBgTx-5-HT3ARs with high affinity (K d = 11 nM), application of nonradioactive αBgTx (up to 300 μM) had no effect on serotonin-induced current responses. αBgTx-5-HT3ARs were purified on an αBgTx-derivatized affinity column from detergent extracts in milligram quantities and at ∼25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-125Iiodophenyl)diazirine (125ITID) was used to identify the amino acids at the lipid−protein interface of purified and lipid-reconstituted αBgTx-5-HT3ARs. 125ITID photoincorporation into the αBgTx-5-HT3AR subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and ∼60% of incorporated 125I, and 17 kDa, containing the M1−M3 transmembrane segments. Within the M4 segment, 125ITID labeled Ser451, equivalent to the 125ITID-labeled residue Thr422 at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) α1M4 α-helix. These results provide a first definition of the surface of the 5-HT3AR M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.
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![[3H]Epibatidine Photolabels...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0021-9258_1067-8816_1083-351X/small "[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors")