Native GI Research Articles

Immunogenicity Characterization of the Recombinant gI Protein Fragment from Pseudorabies Virus and an Evaluation of Its Diagnostic Use in Pigs.

Serological testing is an important method for the diagnosis of pseudorabies virus (PRV) infection. We aimed to investigate the envelope glycoprotein I (gI) of PRV, a strong immunogen, and its potential as an efficient and low-cost diagnostic reagent. In this study, the DNA of the PRV SC strain was used as the template, and the recombinant fragment of gI (633 bp) was amplified via PCR using synthetic primers, and was then ligated into the pET-30a expression vector. The constructs were transferred into Escherichia coli (E. coli) for prokaryotic expression, and the antigenicity of the expression products was identified by Western blot analysis with pig positive serum against PRV. The recombinant protein was purified by a Ni column, and BALB/c mice were immunized with purified gI protein to obtain anti-gI-positive serum. After PK-15 cells had been infected by PRV for 48 h, the immunogenicity of purified gI protein was identified with a fluorescence immunoassay using anti-gI mouse serum. The recombinant plasmid (pET-30a-gI) was expressed, and the native gI protein was obtained after denaturation by urea and renaturation by dialysis. A small-scale ELISA test containing 1.0 µg/mL of purified gI protein was designed to evaluate pig serum (80 samples), and the results of the ELISA test were compared to those of competitive ELISA (cELISA) tests using IDEXX Kits, which resulted in 97.5% consistency. The results suggested that the truncated gI protein may be a potential diagnostic reagent.

MASS CYTOMETRY ANALYSIS OF CROHN’S DISEASE-LIKE PHENOTYPE OF THE ILEOANAL POUCH FOLLOWING ILEAL POUCH-ANAL ANASTOMOSIS FOR ULCERATIVE COLITIS

Abstract BACKGROUNDS For patients with medically refractory ulcerative colitis (UC), total proctocolectomy and ileal pouch-anal anastomosis (IPAA) has been the definitive surgery of choice. Approximately 10% of patients who were initially diagnosed with UC and underwent an IPAA, subsequently develop a condition referred to as Crohn’s disease-like phenotype of ileoanal pouch (CDP). CDP is typified by ulcers in the pre-pouch ileum, stricture involving the pouch or pre-pouch ileum, and/or fistulas. The etiology of CDP remains unknown and remains difficult to diagnose and treat. To define the mucosal immunology of CDP, we analyzed ileal samples from patients with CDP and patients with a normal pouch and pre-pouch ileum using mass cytometry (CyTOF). METHODS Ileal mucosal biopsies from patients with a history of CDP with long-segmental pre-pouch ileitis (n=10) and those with a normal pouch and pre-pouch ileum after IPAA for UC (n=9) were collected during routine pouchoscopy, frozen in medium, and processed, barcoded and analyzed in one CyTOF experiment. Data were analyzed by using viSNE for dimensionality reduction and CITRUS for clustering in Cytobank. RESULTS Compared with patients with a normal pouch/ileum, we observed the following in ileal samples from CDP based on viSNE analysis (Figure 1): 1) A decreased abundance of CD8+CD103+ tissue-resident T cells (TRM). This result is similar to data from colonic mucosal samples in patients with UC (our internal data not shown here and others), but unlike data from prior studies derived from inflamed ileal mucosal specimens in patients with CD (native bowel anatomy without IPAA) which had increased CD8+ TRM. 2) An increase in CD4+CD103-CD161+ Th17 cells previously linked to an activated Th17 phenotype in Crohn’s disease specimens. 3) Increased CD4+CD103-CD45RA+ T cells associated with reduced IL-10/IL-4 and increased TNF-a in IBD. 4) A higher abundance of CD4+CD103-TIGIT+ T cells that were previously shown to predict severity of pediatric IBD. 5) A decrease in CD8+CD103+CD127+ TRM in CDP, although its role in IBD is unclear. The findings from viSNE were confirmed by CITRUS (Figure 2). Further analyses of pouch samples as well as ileal samples from UC and CD with native GI anatomy (for comparison) using CyTOF and scRNAseq are currently underway. CONCLUSIONS We used mass cytometry to identify immune cells specific to ileal mucosa from patients with CDP. Interestingly, immune cells from the inflamed pre-pouch ileum share some features of those from either UC or CD with native bowel anatomy. We also identified an altered immune cell population which appears to be specific to CDP. These data reveal a unique immune landscape of CDP which may serve as a foundation for identifying therapeutic approaches that target specific cell populations for medical management of CDP.

553 Clinical characteristics of acute graft-versus-host disease in small bowel and multi-visceral transplant recipients

Acute graft-versus-host disease (aGVHD) following stem cell transplantation is characterized by rash, liver dysfunction, and diarrhea. Although aGVHD is uncommon following solid organ transplantation, we find higher rates of aGVHD after intestinal (ITx) and multi-visceral transplantation (MVT). In these cases we find a unique presentation as the donor liver (if present) and intestine are spared, leaving skin as the main target. Between 2013 and 2019, we diagnosed 17 cases of aGVHD following ITx (n=9) and MVT including both intestine and liver (n=8). Median time to onset of aGVHD was 37 days. Erythema on the trunk, palms, or both was the most common presenting skin finding (15/17 patients). All patients had skin biopsies at the time of diagnosis. Patients presented with grade 1 (7/17), grade 2 (8/17), grade 3 (1/17), and grade 4 (1/17) aGVHD. Peripheral blood chimerism studies obtained in 11 patients revealed donor CD3+ lymphocytes in 5 (45%). Two patients had passenger lymphocyte syndrome, and partial donor stem cell engraftment was diagnosed in a third patient. Native GI tract involvement (usually rectosigmoid), was found in 7/17 patients; other sites included bone marrow, and native liver. In all, 6/17 patients had no evidence of extra-cutaneous aGVHD. Treatment strategies included systemic and topical corticosteroids, anti-thymocyte globulin, extracorporeal photopheresis, and/or JAK inhibitors. Complications of immunosuppression including opportunistic infections (aspergillosis and toxoplasmosis) and post-transplant lymphoproliferative disorder occurred in 7/17 patients. In summary, rash is typically the first, and may be the only, manifestation of aGVHD following luminal organ transplants. Diagnosis may be aided by time of onset of cutaneous manifestations, extra-cutaneous signs, and peripheral blood chimerism.

1057 EUS-GUIDED DUODENO-JEJUNOSTOMY (EUS-DJ) WITH LUMEN APPOSING METAL STENT (LAMS) AS SALVAGE OPTION FOR PALLIATION OF GASTRIC OUTLET OBSTRUCTION (GOO)

Endoscopic palliation of GOO can be accomplished by either placement of duodenal self-expandable metal stent (SEMS) or endoscopic ultrasound guided gastroenterostomy (EUS-GE). We describe EUS-DJ as a novel salvage endoscopic approach in a patient with native GI anatomy in whom attempts at both SEMS placement and EUS-GE were unsuccessful.

Graft Versus Host Disease Following Intestine Transplantation

Introduction: Graft versus host disease (GVHD) remains one of the most feared medical complications following intestine transplantation (ITx). Few contemporary studies have evaluated the incidence and outcomes of GVHD following ITx. Methods: All ITx performed at our institution from 2003 through 2016 were included in this study and evaluated with the use of a prospectively maintained database. The incidence, associated clinical factors, treatment and outcomes were evaluated for all patients with GVHD. Results: There were 234 transplants performed in 228 patients during this period, including 133 isolated intestine, 59 liver-intestine, 35 multivisceral and 7 modified multivisceral transplants. There were 13 (5.6%) cases of GVHD (6 adults and 7 pediatric) with a median presentation of 77 days (range 14–373) following transplantation. 8 patients received multivisceral or modified multivisceral grafts, 4 patients received isolated intestine grafts, and 1 liver-intestine. 8 patients had native splenectomy at time of transplant and 3 patients had a known immunodeficiency. The most common presentation was a rash (n=8), other clinical manifestations included native GI involvement (n=7), pulmonary involvement (n=3), and bone marrow involvement (n=3). Chimerism data was available for 8 patients and was positive in 7. Immunosuppression was augmented in all cases and median number of treatments/patient was 2 (range 1–4). Treatments included steroids (n=13), thymoglobulin (n=6), alemtuzumab (n=2), extracorporeal photopheresis (n=3), and bone marrow transplant (n=1). The majority of cases 9/13 (69.3%) were fatal despite aggressive treatment. The 5-year survival for patients with GVHD was 30.7% versus 70.5% for patients without GVHD (p=0.001). Statistically significant risk factors for GVHD included multivisceral graft and native splenectomy. Only 1 (7.6%) patient with GVHD had acute rejection compared to 17.7% in the patients without GVHD (p=0.001). Conclusions: Despite modern immunosuppression regimens and advances in treatment, GVHD carries a high mortality. New therapies for GVHD are needed as well as aggressive surveillance protocols in multivisceral graft recipients and patients with a history of splenectomy.

Graft-versus-host disease in paediatric solid organ transplantation: A review of the literature.

GvHD is a rare and serious complication of organ transplantation. The literature is sparse following solid organ transplantation. The aim of this report was to review the literature of GvHD in paediatric solid organ transplantation. We searched PubMed for English-language full-text manuscripts between 1990 and 2015 for eligible studies. A total of 28 publications were found pertaining to paediatric GvHD following solid organ transplantation. GvHD had a mean incidence of 11% (range 8.3-13.4%) following SBTx and 1.5% following liver transplantation. Where described, the most common sites for presentation of GvHD were the skin (87%), the native GI tract (43%), the lungs (7%), the eyes (4%), HA (4%), and the kidneys (1%). Diagnosis was confirmed with biopsy (93%) and/or chimerism (41%). Treatments used include steroids (80%), of which 75% showed partial or complete resolution. Mortality was 33.3% (range 0-100%). Novel therapies include ECP and MSC therapy. GvHD is a rare but serious disease with high mortality. Novel therapies may offer hope in the future, but currently there is limited evidence for their efficacy in the context of intestinal or liver transplantation.

Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80°C of 59.58U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90°C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20h at 80°C in the absence of substrate). Its optimum pH was around 7.0, and it had 86% of its maximum activity at pH 6.0 incubated for 12h at 60°C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

Prophylactic and Pre-Emptive Therapies Using Ganciclovir and CMV Immunoglobulin Result in a Significant Reduction of CMV Disease after Intestinal Transplantation

Introduction: CMV has plagued the field of intestinal transplantation (ITx). The combination of heavy immunosuppression in pediatric recipients of viral trophic transplanted organs has resulted in CMV disease rates reported between 10-35%. Significant graft and patient loss has also been reported. The paucity of recent data on this topic as well as the severity of the problem, prompted this retrospective review. Materials and methods: An IRB approved, retrospective review of a prospectively maintained database was undertaken at a single transplant center. All ITx recipients between 1991-2011 were included. Standard demographic data was collected. End points were survival and CMV disease defined as biopsy proven tissue invasive infection. Since 1996, a strict protocol of prophylaxis against CMV was instituted (TABLE). At the same time, a pre-emptive protocol was initiated using the results of peripheral blood monitoring for the presence of CMV DNA detected by PCR. Samples were collected every 7-30 days and pre-emptive therapy was initiated for positive results (TABLE). Surveillance endoscopy and biopsy was undertaken at predetermined intervals; tissue biopsies were also undertaken when organ dysfunction was detected clinically or on laboratory testing.Table: [Protocol]Results: 100 patients received 119 ITx. The majority were male (60%), Hispanic (54%), children (73%). Overall patient and graft survival at 1 year was 80% and 76%. High risk combinations defined by donor and recipient serologic status at the time of ITx occurred in 73%. All but the first 5 ITx received the protocol. Eight episodes (6.5%) of CMV disease occurred in 7 patients at a mean of 699 days after ITx (131-1791d). Affected tissues were native lung (n=2), native GI tract (n=3), transplanted liver (n=2), and transplanted intestine (1). No graft losses occurred due to CMV. One patient with concomitant underlying immunodeficiency disorder died directly as a result of CMV pneumonitis. CMV free survival was 99% at 1 year. Discussion: This study represents one of the few large single center reports on CMV after ITx. Using a protocol of prophylactic and preemptive therapies directed against CMV, a remarkably low rate of tissue invasive disease was seen. No tissue invasive disease was identified during the prophylactic period. In recipients without immunodeficiency, no patient or graft loss due to CMV was seen. We recommend use of such protocols to reduce CMV disease rates and improve patient and graft survival after ITx.

Influence of Disulfide Connectivity, Electrostatics, and Hydrophobicity on the Conformational Variations of α-Conotoxin GI Single-Disulfide Analogues: Simulations with Polarizable Force Field

The roles of the disulfide bridge, electrostatics, and hydrophobic/hydrophilic effects in the structural stability and conformational changes of six single-disulfide analogues of alpha-conotoxin GI(2-7;3-13) in aqueous solution are investigated by using molecular dynamics simulations with a fragment-based polarization model (J. Phys. Chem. A 2008, 112, 9854.). It is found that the relative stabilities are largely determined by the dipole-dipole interactions between secondary structure-based fragments, revealing the favorable effect of polar residues on conformational stabilities. The loop size closely correlates to not only the thermodynamic stability but also the local geometry of disulfide bridge. The disulfide loops with no more than five residues [GI(2-7), GI(3-7), and GI(7-13)] choose the left-handed disulfide conformation, while the larger loops [with nine and 10 residues in GI(3-13) and GI(2-13)] and a smaller disulfide loop [GI(2-3) without intercysteine residue] prefer the right-handed configuration. In the left-handed analogues, the dihedral angles concerning disulfide bonds decrease subtly along with the enlargement of disulfide loops. A converse dihedral angle and loop size relationship is found in the right-handed isomers. These results are rationalized by the strain energy of the disulfide bond as well as the electrostatic and van der Waals interactions between cysteine pairs. The single-disulfide analogues also exhibit much higher conformational diversity than the native GI. The important role of the size of hydrophobic core in the conformational evolution is also demonstrated in terms of the radius of gyration of the hydrophobic region. The radial distribution functions show the significant solvent-solute hydrogen bonding, implying that the interplay between the intermolecular and the intramolecular interactions control the dynamic process of GI single-disulfide analogues.

Quantification of Gi-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y14 Receptor

The P2Y14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162-168, 2009). In the current study, we examined P2Y14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y14 receptor.

Conformational Simulations of Aqueous Solvated α-Conotoxin GI and Its Single Disulfide Analogues Using a Polarizable Force Field Model

The solution conformation of alpha-conotoxin GI and its two single disulfide analogues are simulated using a polarizable force field in combination with the molecular fragmentation quantum chemical calculation. The polarizability is explicitly described by allowing the partial charges and fragment dipole moments to be variables, with values coming from the linear-scaling energy-based molecular fragmentation calculations at the B3LYP/6-31G(d) level. In comparison with the full quantum chemical calculations, the fragmentation approaches can yield precise ground-state energies, dipole moments, and static polarizabilities for peptides. The B3LYP/6-31G(d) charges and fragment-centered dipole moments are introduced in calculations of electrostatic terms in both AmberFF03 and OPLS force fields. Our test calculations on the gas-phase glucagon (PDB code: 1gcn) and solvated alpha-conotoxin GI (PDB code: 1not) demonstrate that the present polarization model is capable of describing the structural properties (such as the relative conformational energies, intramolecular hydrogen bonds, and disulfide bonds) with accuracy comparable to some other polarizable force fields (ABEEM/MM and OPLS-PFF) and the quantum mechanics/molecular mechanics (QM/MM) hybrid model. The employment of fragment-centered dipole moments in calculations of dipole-dipole interactions can save computational time in comparison with those polarization models using atom-centered dipole moments without much loss of accuracy. The molecular dynamics simulations using the polarizable force field demonstrate that two single disulfide GI analogues are more flexible and less structured than the native alpha-conotoxin GI, in agreement with NMR experiments. The polarization effect is important in simulations of the folding/unfolding process of solvated proteins.

Synthesis and immunological studies of alpha-conotoxin chimera containing an immunodominant epitope from the 268-284 region of HSV gD protein.

We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrophilic and adopts a beta-turn. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, stabilized by two disulfide bridges at positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, 8Arg-His-Tyr-Ser12 has been replaced by Asp-Pro-Val-Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3-13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys2-Cys7 three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 M HCl or Tl(tfa)3 in trifluoroacetic acid (TFE)] were compared. The high-performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha-[Tyr1]-conotoxin chimeric peptide and native alpha-conotoxin GI showed similar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type antibody responses showed that the bicyclic HSV-alpha-[Tyr1]-conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV-alpha-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha-[Tyr1]-conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.

Structure determination of the three disulfide bond isomers of α-conotoxin GI: a model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of α-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. α-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2–7;3–13), GI(2–13;3–7) and GI(2–3;7–13). The NMR structure for the native isomer GI(2–7;3–13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Å for a family of 35 structures, and comprises primarily a distorted 310helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2–7;3–13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in α-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2–7;3–13)<GI(2–13;3–7)<GI(2–3;7–13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2–3;7–13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2–7;3–13) to the mammalian nAChR is proposed, with an α-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the α and δ subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2–7;3–13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.

G protein binding and G protein activation by nucleotide transfer involve distinct domains on tubulin: regulation of signal transduction by cytoskeletal elements.

Cytoskeletal proteins have been demonstrated to interact with elements which mediate neuronal signal transduction. This laboratory has shown that tubulin forms complexes with certain G proteins and transfers GTP to them, and such nucleotide transfer correlates well with the inhibition of adenylyl cyclase. A previous study showed that tubulin-G protein complex formation occurred at regions of tubulin which were likely to be involved in binding to other tubulin dimers during the process of microtubule polymerization [Wang, N., & Rasenick, M. M. (1991) Biochemistry 30, 10957-10965]. This study represents an attempt to investigate the regions of tubulin which orchestrate the transfer of GTP from tubulin to G protein. It is demonstrated that two monoclonal antibodies, with determinants in similar regions of alpha (DM1a) or beta (DM1b) tubulin, blocked (by 70-80%) the ability of tubulin dimers (with GppNHp bound) to promote a stable inhibition of adenylyl cyclase. Under the same conditions, a polyclonal antitubulin antibody caused only a slight reduction in adenylyl cyclase inhibition. None of the antibodies altered the inhibition of adenylyl cyclase induced by GppNHp in the absence of tubulin. Under conditions where tubulin-GppNHp inhibited synaptic membrane adenylyl cyclase, tubulin dimers with the photoaffinity GTP analog azidoanilido-GTP (32P-AAGTP) bound transferred that nucleotide to G alpha i1 on the membrane. DM1a and DM1b blocked this nucleotide transfer. Similarly, tubulin-AAGTP transferred AAGTP to purified G proteins in solution and DM1a and DM1b blocked this process as well. Despite their ability to block the activation of Gi1 by tubulin, neither antibody altered the ability of tubulin to bind to native Gi alpha affixed to nitrocellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactivity, epitope mapping and protection studies with anti-conotoxin GI sera and various conotoxins

Goat or rabbit anti-conotoxin GI sera recognized native or dithiothreitol-reduced α-conotoxins GI, MI and SI in an enzyme-linked immunosorbent assay (ELISA). Native μ-conotoxin GIIIA or ω-conotoxin GVIA did not react with either anti-conotoxin GI serum in the assay. The goat anti-conotoxin GI serum neutralized 2.5 ld 50s of α-conotoxins GI or MI in mouse lethal assays, while the rabbit antiserum had little protective capabilities. Epitope mapping of synthesized conotoxin peptide fragments revealed that both anti-conotoxin GI sera recognized linear sequences from five different α-conotoxins: GI, GIA, GII, MI and SI. The CCNPAC sequence was optimally recognized by both antisera.

Primary and secondary structure of conotoxin GI, a neurotoxic tridecapeptide from a marine snail

To determine the mode of disulphide bond formation in conotoxin GI, a tridecapeptide amide with 4 Cys residues, all 3 of its peptides having different modes of disulphide-bond formation were synthesized by solution procedure using selectively removable protective groups at the Cys residues. After deprotection with HF, one pair of acetamidomethyl groups was left unremoved, and then two sets of disulphide bonds were formed selectively. The toxic potency in mice of one product was comparable with that reported for native conotoxin GI and was almost 10-fold as high as that of the other two products. The toxicity of the native toxin reportedly is not regenerated upon reduction and reoxidation, but this study showed that the most toxic product was the most readily formed one.