Somatic Mutations During an Immune Response in Xenopus Tadpoles

The tadpole B-cell repertoire is less diverse than that of the adult frog; their antibodies are of lower affinity and are less heterogenous. In order to determine whether this difference is due to a lack of or a reduced rate of somatic hypermutation, we analyzed and compared cDNA sequences utilizing VH1 elements with germline counterparts in isogenic LG7 tadpoles during an immune response. Indeed, tadpole VH1 sequences contained somatic mutations. There were zeo to 5 mutations per sequence, all single base-point mutations, with the high ratio of GC to AT base-pair alterations similar to that observed in adult frogs

Sequences of Cμ and the VH1 Family in LG7, a Clonable Strain of Xenopus, Homozygous for the Immunoglobulin Loci

Twenty-eight heavy-chain variable (VH1) region genes and the immunoglobulin (IgM) heavy-chain constant region of an isogenic Xenopus hybrid, X. laevis/X, gilli, LG7, have been sequenced. The LG7 clone represents the first Xenopus hybrid that is homozygous for the IgH locus. The VH1 family was specifically investigated because VH1 genes are used by the antibodies produced during the Xenopus antidinitrophenol (DNP) response, These VH1 germ-line sequences establish a so-called ”dictionary— that is available for studying somatic hypermutational mechanisms in immunized frogs

The clinical features of juvenile dermatomyositis: A single-centre inception cohort

Introduction: Juvenile Dermatomyositis (JDM), a severe and rare autoimmune disease, is the most common idiopathic inflammatory myopathy in children. We describe the clinical features of a large single-centre cohort.Methods: We studied an inception cohort (0-18 years old) referred for diagnosis to the JDM clinic at The Hospital for Sick Children (SickKids), between January 1989 and September 2017. Probable or definite diagnosis of JDM was done according to the 2017 ACR/EULAR Criteria. We excluded children who had treatment started at another hospital. The data were collected retrospectively from clinical charts and the SickKids JDM database.Results: 172/230 (74.8%) patients were included. They were most often female (female:male = 1.8:1); the age at diagnosis was 8.5 +/- 4.3 years. There was a positive family history for autoimmune disease in 52%, mainly rheumatoid arthritis. No patient died. The most common signs at inception were muscle weakness (85.5%), nailfold capillary abnormalities (83.4%), Gottron papules (78.5%), heliotrope rash (66.3%), abnormal gait (55.8%), and malar/facial rash (54.7%). The prevalence of Gottron papules, heliotrope rash, facial/malar rash, nailfold capillary abnormalities, Raynaud phenomenon, dysphonia/dysphagia (a frequent cause of hospitaliza-tion), mouth ulcers, calcinosis, eye problems, joint involvement, acanthosis nigricans and lipodystrophy increased during follow-up. Muscle enzymes, namely CK, ALT, AST, were often normal or only slightly raised despite active muscle disease; conversely LD was often high. Anti-Nuclear Autoantibodies were positive in 49.7% of patients at diagnosis. The course of the disease was: 29.1% monocyclic, 5.3% polycyclic, 33.1% chronic. The course of 56 patients (32.5%) was not classifiable due to length of follow-up. Corticosteroids were used as treatment in almost all our patients and 30% required intravenous therapy due to the severity of the presen-tation; methotrexate was added in 64%, more often in recent years. Unresponsive patients were treated mostly with intravenous immunoglobulins (IVIG).Conclusions: The information obtained from this relatively large number of patients adds to the growing knowledge base of this rare disease. Trial registration: SickKids Research Ethics Board approved the study

Me-Talnetant and Osanetant Interact within Overlapping but Not Identical Binding Pockets in the Human Tachykinin Neurokinin 3 Receptor Transmembrane Domains

ABSTRACT Recent clinical trials have indicated that neurokinin 3 receptor antagonists (S)-(ϩ) - 6.51 F, although not affecting Me-talnetant, led to a significant decrease in affinity and potency of osanetant. The mutation N142 2.61 A, which abolished the potency and affinity of osanetant, led to a significant increase in the affinity and potency of Me-talnetant. The proposed docking mode was further validated using (S)-2-(3,5-bis-trifluoromethyl-phenyl)- N-[4-(4-fluoro-2-methyl-phenyl)-6-((S)-4-methanesulfonyl-3-methyl-piperazin-1-yl)-pyridin-3-yl]-N-methyl-isobutyramide (RO49085940), from another chemical class. It is noteworthy that the mutation F342 7.39 A caused an 80-fold gain of RO4908594 binding affinity, but the same mutation resulted in the complete loss of the affinity of Me-talnetant and partial loss of the affinity of osanetant. These observations show that the binding pocket of Me-talnetant and osanetant are overlapping, but not identical. Taken together, our data are consistent with the proposed docking modes where Me-talnetant reaches deeply into the pocket formed by transmembrane (TM)1, -2, and -7, whereas osanetant fills the pocket TM3, -5, and -6 with its phenyl-piperidine moiety. The tachykinin family comprises the neuropeptides substance P (SP; RPKPQQFFGLM-NH2), neurokinin A (NKA; HKTDSFVGLM-NH2), and neurokinin B (NKB; DMHD-FFVGLM-NH2), which share a common C-terminal sequence, FXGLM-NH2. SP, NKA, and NKB act as neurotransmitters or neuromodulators, and they elicit their effects through three types of neurokinin receptors: NK 1 R, NK 2 R, and NK 3 R, respectively. Neurokinin receptors (NKRs) belong to the superfamily of G protein-coupled receptors (GPCRs) that couple via G q/11 to the activation of phospholipase C, leading to elevation of intracellular Ca 2ϩ levels ABBREVIATIONS: SP, substance P; NK, neurokinin; NKR, neurokinin receptor; GPCR, G protein-coupled receptor; 3D, three-dimensional; Me-talnetant, (S) IP, inositol phosphates; h, human; TM, transmembrane; EC2, extracellular loop 2; I3, intracellular loop 3; HEK, human embryonic kidney; RT, room temperature; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary; WT, wild type; HBSS, Hanks' balanced salt solution; 7TMD, seventransmembrane domain; SAR, structure-activity relationship(s); 3D, three-dimensional; AVP, arginine vasopressin; V 1 R, vasopressin 1 receptor. 0026-895X/08/7306-1736 -1750$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 6 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 42754/3336330 Mol Pharmacol 73:1736-1750, 2008 Printed in U.S.A. - 1736 have been implicated in the pathology of psychiatric diseases such as depression, schizophrenia, and anxiety as well as other conditions, including asthma, pain, emesis, and arthritis Although talnetant clearly displayed a reversible and competitive mode of antagonism in the NKB-induced Ca 2ϩ mobilization at cloned hNK 3 R and in the senktide-induced contractions in rabbit isolated iris sphincter muscles 3 H]Me-talnetant and [ 3 H]osanetant bindings, [MePhe 7 ]NKB-stimulated IP formation, and Schild and kinetic analyses to identify the important molecular determinants of NKB, Me-talnetant, and osanetant binding pockets and to elucidate the antagonism mode of both antagonists on wild-type and mutated hNK 3 Rs. Furthermore, these experimental findings allow the construction of a homology model based on the X-ray crystal of bovine rhodopsin Materials and Methods Materials Construction of Point-Mutated hNK 3 Rs. cDNA encoding the human NK 3 R (accession no. P29371) was subcloned into pCI-Neo expression vectors (Promega, Madison, WI). All point-mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions and using pCI-Neo-hNK 3 R as a DNA template. Complementary oligonucleotide primers (sense and antisense) containing the single site or double sites of mutations were synthesized by Microsynth AG (Balgach, Switzerland). The following polymerase chain reaction conditions were used for repeated extensions of the plasmid template: 95°C for 1 min and 20 cycles of 95°C for 30 s, 55°C for 1 min, and 68°C for 8 min using 50 ng of plasmid DNA, 100 ng of each primer, and 2.5 units of Pfu Turbo DNA polymerase (Stratagene). The entire coding regions of all positive point-mutants were sequenced from both strands using an automated cycle sequencer (Applied Biosystems, Foster City, CA). Cell Culture, Large-Scale Transient Transfection, and Membrane Preparation. Human embryonic kidney (HEK) 293-EBNA cells (Invitrogen, Carlsbad, CA) were adapted to grow in suspension in spinner flasks at 95 to 105 rpm. For transfection experiments, we used a modified medium (DHI special; Invitrogen) and for the gene delivery, we used transfection reagent X-tremeGENEQ 2 (Roche Applied Science, Rotkreuz, Switzerland), which consists of substances A and B. Cells were cultured to a density of 6 to 10 ϫ 10 5 cells/ml, centrifuged for 3 min at 600 rpm, and resuspended in DHI media. The cell density was adjusted to 5 ϫ 10 5 cells/ml, and the culture was incubated for at least 3 h before transfection. The transfection complexes were generated in 1/10 of the culture volume in DHI media at room temperature (RT). For 1 ml of culture, first 0.4 g of DNA was added to 0.1 ml of medium, mixed, after 2 min followed by 0.15 g of X-tremeGENEQ A, mixed, and followed after a further 2 min by 0.5 g of X-tremeGENEQ B. The mixture was incubated for 15 min at room temperature to allow DNA complex formation before it was added to the cells. Forty-eight hours after transfection, the cells were harvested and washed three times with ice-cold PBS, and then they were frozen at Ϫ80°C. The pellet was suspended in ice-cold 50 mM Tris, pH 7.4, buffer containing 10 mM EDTA (10ϫ volume) and homogenized with a Polytron homogenizer (Kinematica AG, Basel, Switzerland) for 30 s at 16,000 rpm. After centrifugation at 48,000g for 30 min at 4°C, the pellet was suspended again in ice-cold 10 mM Tris, pH 7.4, buffer containing 0.1 mM EDTA (10ϫ volume), homogenized, and spun again as described above. The pellet was resuspended in ice-cold 10 mM Tris, pH 7.4, buffer containing 0.1 mM EDTA and 10% sucrose (5ϫ volume). After homogenization for 15 s at 16,000 rpm, the protein content was measured using the bicinchoninic acid method (Pierce, Socochim, Lausanne, Switzerland), with bovine serum albumin as the standard. The membrane homogenate was frozen at Ϫ80°C before use. [ 3 H]Me-talnetant and [ 3 H]Osanetant Bindings. After thawing, the membrane homogenates were centrifuged at 48,000g for 10 min at 4°C, the pellets were resuspended in the binding buffer (50 mM Tris-HCl, 4 mM MnCl 2 , 1 M phosphoramidon, and 0.1% bovine serum albumin at pH 7.4) to a final assay concentration of 5 g of protein/well. Saturation isotherms were determined by addition of various concentrations of [ 3 H]Me-talnetant (0.005-10 nM) or [ 3 H]-osanetant (0.009-3 nM) to these membranes (in a total reaction volume of 500 l) for 75 min at RT. At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% polyethylenimine ϩ 0.3% bovine serum albumin; PerkinElmer Life and Analytical Sciences, Waltham, MA) with a FilterMate-96 harvester (PerkinElmer Life and Analytical Sciences) and washed four times with ice-cold 50 mM Tris-HCl, pH 7.4, buffer. Nonspecific binding was measured in the presence of 10 M SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a TopCount microplate scintillation counter (PerkinElmer Life and Analytical Sciences) with quenching correction after addition of 45 l of MicroScint 40 (PerkinElmer Life and Analytical Sciences) and shaking for 1 h. Saturation experiments were analyzed by Prism 4.0 (GraphPad Software Inc., San Diego, CA) using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 M). IC 50 values were derived from the inhibition curve, and the affinity constant (K i ) values were calculated using the Cheng-Prusoff equation is the concentration of radioligand, and K d is its dissociation constant at the receptor, derived from the saturation isotherm 3 H]osanetant. Binding kinetics parameters, K ob and K off values (observed on and off rates, respectively), were derived from association-dissociation curves using the one-phase exponential association and decay equations (Prism 4.0), respectively. K on , half-life and K d were calculated using the K on ϭ (K ob Ϫ K off )/[ligand], t 1/2 ϭ ln2/K, and K d ϭ K off /K on equations, respectively. Statistical significance was determined using a two-tailed t test (Prism 4.0). [ 3 H]Inositol Phosphates Accumulation Assay. [ 3 H]Inositol phosphates (IP) accumulation was measured as described previously Residue Numbering Scheme. The position of each amino acid residue in the seven transmembrane domain (7TMD) was identified both by its sequence number and by its generic numbering system proposed by Alignment and Model Building. The amino acid sequences of the human NK 3 R (accession no. P29371), rat NK 3 R (accession no. P16177), mouse NK 3 R (accession no. P47937), gerbil NK 3 R (accession no. AM157740), human NK 1 R (accession no. P25103), and human NK 2 R (accession no. P21452) were retrieved form the SwissProt database. These amino acid sequences were aligned to the sequence of bovine rhodopsin (accession no. P02699) using the ClustalW multiple alignment program (http://www.ebi.ac.uk/ clustalw/). A slow pairwise alignment using the BLOSUM matrix series Using this alignment and the X-ray structure of bovine rhodopsin Results talnetant concentrations of 0.005-10 nM and [ 3 H]osanetant concentrations of 0.009-3 nM) and best fitted to a one-site model for both radioligands Mapping the Binding Pocket of NK 3 Receptor Ligands 1739 the binding modes of Me-talnetant and osanetant, an alignment of the seven transmembrane helices of the whole NK family toward the transmembrane helices of bovine rhodopsin (Protein Data Bank reference code 1f88) was made. The inverse agonist of rhodopsin, 11-cis-retinal, was used as a template for the locations of Me-talnetant and osanetant. Amino acids, which were found 6.0 Å away from retinal in the X-ray crystal structure of rhodopsin Subsequently, one of our aims was to address the selectivity of Me-talnetant and osanetant for NK 3 R over NK 1 R and NK 2 R. Although it was not possible to generate all possible mutations of interest, we have chosen a subset of 12 mutants to obtain the information to learn which residues influence selectivity. According to the predicted docking poses, the residues at position 3.36, 6.51, and 7.39 that seem to be important factors for the selectivity of Me-talnetant and osanetant were mutated to the respective residues in hNK 1 R or hNK 2 R (V169M, Y315F, and F342M). Phe342 was additionally mutated to alanine, a decision based on the observation that the 4-phenyl substituent of RO4908594 seems to clash into Phe342. Hence, F342A could result in an affinity increase for this compound, an essential test in the validation of the docking poses. NK 1 R is not only characterized by having a methionine in position 7.39 but also the neighboring residue 7.38 differs from NK 3 R (serine in NK 3 R, isoleucine in NK 1 R). Therefore, the double-mutant S342I/F342M was also generated to correctly introduce the NK 1 R region in NK 3 R. The residue at position 7.38 is too far away from the ligands to make a direct interaction with Me-talnetant or osanetant. Nevertheless, although Ser341 is not predicted to be in direct contact with the docked ligands, it could cause a kink of the helix TM7 as a result of its hydrogen-bonding capabilities. Thus, an additional mutant, S341A, was generated. For the case where this mutation would influence Me-talnetant or osanetant binding or the above-mentioned double-mutant would point to an influence of position 7.38 onto binding, single point-mutations S341I (NK 1 R) and S342L(NK 2 R) would have to be tested because they could indirectly influence binding by a change of TM7 helix conformation. But these mutations will be no longer be needed in case that S341A does not change the binding affinities. Comparison ]-NKB, the mutants N138A, L232A, Y315A, and M346A showed 59.4-, 10.0-, 6.2-, and 14.1-fold (statistical significance of P ϭ 0.0009, P ϭ 0.0002, P ϭ 0.02, and P ϭ 0.006), respectively, lower affinity for [MePhe 7 ]NKB than the WT 3 H]osanetant competition binding assay. RO4908594 is a potent dual NK 1 R/NK 3 R antagonist that binds to hNK 1 R, hNK 2 R, and hNK 3 R, with K i values of 0.8 Ϯ 0.1, Ͼ10,000, and 1.6 Ϯ 0.1 nM, respectively Mapping the Binding Pocket of NK 3 Receptor Ligands 1741 TABLE 2 Effects of the mutations on ͓ 3 H͔Me-talnetant displacement by ͓MePhe 7 ͔NKB, osanetant, and SB222200 in the membrane preparations from HEK293-EBNA cells transiently expressing WT and mutated hNK 3 Rs K i and n H values for ͓ 3 H͔Me-talnetant binding inhibition by ͓MePhe 7 ͔NKB, osanetant, or SB222200 were calculated as described under Materials and Methods. Values are mean Ϯ S.E. of the K i calculated from three independent experiments, each performed in duplicate. The mutations that affected the affinity constants of ͓MePhe 7 ͔NKB, osanetant, and SB222200 in comparison with WT are shown in boldface type. Statistical significance was determined using a two-tailed t test. [MePhe 7 ]NKB have been measured in the presence of 0, 10, 30, and 100 nM Me-talnetant or 0, 30, 100, and 300 nM osanetant in CHO cells expressing transiently the WT and mutated hNK 3 Rs. As seen in Mapping the Binding Pocket of NK 3 Receptor Ligands 1743 As observed above The dissociation rates for [ 3 H]Me-talnetant and [ 3 H]osanetant binding to the WT receptor was determined by the addition of an excess amount of SB222200 after equilibrium (30 min and 1 h, respectively) was reached. The reversal of binding for both antagonists was complete, with t 1/2 values of 4.6 and 10 min, respectively ( 4.9 Ϯ 0. Docking of Me-Talnetant, Osanetant, and RO4908594 onto the NK 3 R-7TMD Binding Cavity. To visualize the mutation data, a 3D model of the hNK 3 R-7TMD using the atomic coordinates of bovine rhodopsin (Protein Data Bank reference code 1f88) was constructed. Mapping the Binding Pocket of NK 3 Receptor Ligands 1745 proposed for RO4908594 The proposed docking pose

Tibia stress injury and the imaging appearance of stress fracture in juvenile dermatomyositis: six patients’ experiences

Abstract Background Tibial stress injuries are frequent injuries of the lower extremity and the most common causes of exercise-induced leg pain among athletes and military recruits. They sometimes occur in patients with pathological conditions of bone metabolism such as osteoporosis or rheumatoid arthritis, but there are previously no cases reported in juvenile dermatomyositis (JDM). Here we report 6 JDM patients who presented with shin pain, and the imaging appearance of tibial stress fractures or stress reactions. Case presentation All 6 patients with JDM presented with shin pain or tenderness in the anterior tibia without any evidence of excessive exercise or traumatic episode. They were diagnosed with tibial stress injuries based on a combination of radiographs, three-phase bone scans, and magnetic resonance imaging (MRI), and 5 out of 6 patients had been treated with prednisone and/or methotrexate at onset of tibial stress injuries. In one patient, we could not find any abnormalities in his radiograph, but the subsequent MRI showed tibial stress reaction. In all 6 patients, the tibial stress injuries improved with only rest and/or analgesics. Conclusion We experienced 6 children with JDM who presented with shin pain, and who were diagnosed with tibial stress fractures or stress reactions. Their underlying disease and weakness, treatment with glucocorticoid and methotrexate, or inactivity may have resulted in these tibial injuries, and made these patients more predisposed than other children. In addition to preventing JDM patients from getting osteoporosis, we need to consider stress reactions when children with JDM complain of sudden shin pain