[Renal transplantation, anxiety and depressive disorders and quality of life]. / Transplantation rénale, troubles anxiodépressifs et qualité de vie.

Until now there are few data in the literature describing psychiatric comorbidity in patients waiting for renal transplantation. We have conducted a cross sectional study estimating the prevalence of anxiety and depressive disorders in three groups of renal transplant patients, before transplantation, six months and one year after. The MINI was used to estimate the prevalence of anxiety and depressive disorders. Anxiety and depressive symptoms were assessed using the HAD. Patients' quality of life was also assessed using the SF-36. This study did not find any major impact of renal transplantation on the prevalence of structured psychiatric disorders. Indeed, the prevalence of depressive and anxiety disorders did not differ significantly between the three groups. The mean scores of anxiety did not differ significantly between the three groups in contrast to the mean scores of depression, which differed significantly between the group "before transplantation" and the group "one year after transplantation". We did not find any significant difference concerning the scores of patient's quality of life between the three groups, except for the item "health perceived by the patients themselves". Health perceived by the patients was greater in the group "after transplantation". The quality of life of dialysed or transplant patients was strongly correlated with anxiety and depressive symptoms scores, emphasizing the major interest of a multidisciplinary approach for these patients.

New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification.

We report the first use of tandem affinity purification (TAP) in a prokaryote to purify native protein complexes, and demonstrate its reliability and power. We purified the acyl carrier protein (ACP) of Escherichia coli, a protein involved in a myriad of metabolic pathways. Besides the identification of several known partners of ACP, we rediscovered ACP/MukB and ACP/IscS interactions already detected but previously disregarded as due to contamination. Here, we demonstrate the specificity of these interactions and characterize them. This suggests that ACP is involved in additional previously unsuspected pathways. Furthermore, this study shows how the TAP method can be simply used in prokaryotes such as E. coli to identify new partners in protein-protein interactions under physiological conditions and thereby uncover novel protein functions.

Isolation of a tarantula toxin specific for a class of proton-gated Na+ channels.

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H(+)-gated Na(+) channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC(50) = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

MIT(1), a black mamba toxin with a new and highly potent activity on intestinal contraction.

Mamba intestinal toxin (MIT(1)) isolated from Dendroaspis polylepis venom is a 81 amino acid polypeptide cross-linked by five disulphide bridges. MIT(1) has a very potent action on guinea-pig intestinal contractility. MIT(1) (1 nM) potently contracts longitudinal ileal muscle and distal colon, and this contraction is equivalent to that of 40 mM K(+). Conversely MIT(1) relaxes proximal colon again as potently as 40 mM K(+). The MIT(1)-induced effects are antagonised by tetrodotoxin (1 microM) in proximal and distal colon but not in longitudinal ileum. The MIT(1)-induced relaxation of the proximal colon is reversibly inhibited by the NO synthase inhibitor L-NAME (200 microM). (125)I-labelled MIT(1) binds with a very high affinity to both ileum and brain membranes (K(d)=1.3 pM and 0.9 pM, and B(max)=30 fmol/mg and 26 fmol/mg, respectively). MIT(1) is a very highly selective toxin for a receptor present both in the CNS and in the smooth muscle and which might be an as yet unidentified K(+) channel.

Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis.

1. In the present study, two new peptides, phrixotoxins PaTx1 and PaTx2 (29-31 amino acids), which potently block A-type potassium currents, have been purified from the venom of the tarantula Phrixotrichus auratus. 2. Phrixotoxins specifically block Kv4.3 and Kv4.2 currents that underlie I(to1), with an 5 < IC50 < 70 nM, by altering the gating properties of these channels. 3. Neither are the Shaker (Kv1), Shab (Kv2) and Shaw (Kv3) subfamilies of currents, nor HERG, KvLQT1/IsK, inhibited by phrixotoxins which appear specific of the Shal (Kv4) subfamily of currents and also block I(to1) in isolated murine cardiomyocytes. 4. In order to evaluate the physiological consequences of the Ito1 inhibition, mice were injected intravenously with PaTx1, which resulted in numerous transient cardiac adverse reactions including the occurrence of premature ventricular beats, ventricular tachycardia and different degrees of atrioventricular block. 5. The analysis of the mouse electrocardiogram showed a dose-dependent prolongation of the QT interval, chosen as a surrogate marker for their ventricular repolarization, from 249 +/- 11 to 265 +/- 8 ms (P < 0.05). 6. It was concluded that phrixotoxins, are new and specific blockers of Kv4.3 and Kv4.2 potassium currents, and hence of I(to1) that will enable further studies of Kv4.2 and Kv4.3 channel and/or I(to1) expression.

The rat dermorphin-like immunoreactivity is supported by an aminopeptidase resistant peptide.

Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.

Production and characterization of site-directed antibodies against dermorphin and dermorphin-related peptides.

To detect and purify endogenous dermorphin-like molecules in mammalian tissues, an immunological approach was developed. Site-directed antibodies against synthetic dermorphin and related dermorphin peptides were produced. The immunogenic forms of dermorphin were selected to obtain antibodies recognizing different epitopes overlapping the whole dermorphin molecule. One of them specifically recognized the crucial "opioid message" (the N-terminal part of the molecule), which is required for a ligand to exert its full opioid activity. The validity of our immunological approach was analyzed by studying the dermorphin-related peptide distribution in Phyllomedusa sauvagei skin. The finding that tetrapeptide Y-A-G-F-OH was present in Phyllomedusa sauvagei extracts suggested that either the Tyr3-Pro6 peptidic bond may be relatively unstable or endogenous proteolytic enzymes present in Phyllomedusa skin may inactivate this peptidic bond.

Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels.

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine.

Structural elements of secretory phospholipases A2 involved in the binding to M-type receptors.

Specific membrane receptors for secretory phospholipases A2 (sPLA2s) have been initially identified with novel snake venom sPLA2s called OS1 and OS2. One of these sPLA2 receptors (muscle (M)-type, 180 kDa) has a very high affinity for OS1 and OS2 and a high affinity for pancreatic and inflammatory-type mammalian sPLA2s, which might be the natural endogenous ligands of PLA2 receptors. Primary structures of OS1 and OS2 were determined and compared with sequences of other sPLA2s that bind less tightly or do not bind to the M-type receptor. In addition, the binding properties of pancreatic sPLA2 mutants to the M-type receptor have been analyzed. Residues within or close to the Ca(2+)-binding loop of pancreatic sPLA2 are crucially involved in the binding step, although the presence of Ca2+ that is essential for the enzymatic activity is not required for binding to the receptor. These residues include Gly-30 and Asp-49, which are conserved in all sPLA2s. Leu-31 is also essential for binding of pancreatic sPLA2 to its receptor. Many other mutations have been considered. Those occurring in the N-terminal alpha helices and the pancreatic loop do not change binding to the M-type receptor. Conversion of pancreatic prophospholipase to phospholipase is essential for the acquisition of binding properties to the M-type receptor.

Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.

Calcicludine (CaC) is a 60-amino acid polypeptide from the venom of Dendroaspis angusticeps. It is structurally homologous to the Kunitz-type protease inhibitor, to dendrotoxins, which block K+ channels, and to the protease inhibitor domain of the amyloid beta protein that accumulates in Alzheimer disease. Voltage-clamp experiments on a variety of excitable cells have shown that CaC specifically blocks most of the high-threshold Ca2+ channels (L-, N-, or P-type) in the 10-100 nM range. Particularly high densities of specific 125I-labeled CaC binding sites were found in the olfactory bulb, in the molecular layer of the dentate gyrus and the stratum oriens of CA3 field in the hippocampal formation, and in the granular layer of the cerebellum. 125I-labeled CaC binds with a high affinity (Kd = 15 pM) to a single class of noninteracting sites in rat olfactory bulb microsomes. The distribution of CaC binding sites in cerebella of three mutant mice (Weaver, Reeler, and Purkinje cell degeneration) clearly shows that the specific high-affinity labeling is associated with granule cells. Electrophysiological experiments on rat cerebellar granule neurons in primary culture have shown that CaC potently blocks the L-type component of the Ca2+ current (K0.5 = 0.2 nM). Then CaC, in the nanomolar range, appears to be a highly potent blocker of an L-subtype of neuronal Ca2+ channels.

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS)

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 40 mM KCl with a potency (K0.5 = 20 nM) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 100 nM) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (K0.5 = 3 nM) and DNP (K0.5 = 3 nM) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cGMP responses to CNP (K0.5 = 10 nM) than to ANP or DNP (K0.5 = 100 nM). Finally, DNP completely prevented the specific 125I-ANP binding to clearance receptors in cultured aortic myocytes with a potency (Kd = 10 nM) that was less than that of ANP (Kd = 0.3 nM). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors.

Scyllatoxin, a blocker of Ca(2+)-activated K+ channels: structure-function relationships and brain localization of the binding sites.

Chemical modifications of scyllatoxin (leiurustoxin I) have shown that two arginines in the sequence, Arg6 and Arg13, are essential both for binding to the Ca(2+)-activated K+ channel protein and for the functional effect of the toxin. His31 is important both for the binding activity of the toxin and for the induction of contractions on taenia coli. However, although its iodination drastically decreases the toxin activity, it does not abolish it. Chemical modification of lysine residues or of Glu27 does not significantly alter toxin binding, but it drastically decreases potency with respect to contraction of taenia coli. The same observation has been made after chemical modification of the lysine residues. The brain distribution of scyllatoxin binding sites has been analyzed by quantitative autoradiographic analysis. It indicates that apamin (a bee venom toxin) binding sites are colocalized with scyllatoxin binding sites. The results are consonant with the presence of apamin/scyllatoxin binding sites associated with Ca(2+)-activated K+ channels. High-affinity binding sites for apamin can be associated with very-high-affinity (less than 70 pM), high-affinity (approximately 100-500 pM), or moderate-affinity (greater than 800 pM) binding sites for scyllatoxin.

Ig repertoire of human polyspecific antibodies and B cell ontogeny.

A total of 463 EBV Ig-secreting clones were derived from embryonic tissues, cord blood, and adult peripheral blood. Subcloning and analysis of the H and K loci (germline vs rearranged DNA status) of 44 primary clones insured clonality in at least 92% of cases. Whatever the cell origin, a somewhat constant proportion of clones (i.e., 11 to 16%) expressed polyspecific antibodies when tested on a panel of nine Ag, including self-Ag. The VH and VK repertoires have been studied using VH1-VH6 and VK1-VK4 family-specific probes. For all EBV clones the VH and VK utilization was similar to that of the normal untransformed population. A correlation was observed between the level of expression and the gene number for VH, whereas a clear distortion appeared for VK. Moreover, the usage pattern of VH and VK families of the polyspecific clones did not significantly differ from that of clones of unknown specificity, suggesting that polyspecificity was not linked to a restricted repertoire.

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.

Rapid expansion of human immunoglobulin repertoire (VH, V kappa, V lambda) expressed in early fetal bone marrow.

We have isolated pre-B- and B-cell clones after transformation by Epstein-Barr virus (EBV) of human fetal bone marrow cells between weeks 8 and 13 of gestation. These clones were characterized for immunoglobulin (Ig) chain synthesis, the status (rearranged versus germ line) of the heavy (H) chain, kappa, and lambda loci, and of the Ig mRNA transcripts by using specific variable (V), VH, V kappa, and V lambda probes covering the almost complete IgV repertoire. Mature B cells could already be identified in the 8-week-old bone marrow, with both kappa and lambda isotypes being present. Nevertheless, at this stage, most of the clones had Xhe characteristics of pre-B cells, as indicated by the presence of mu transcripts (either functional or sterile) in the absence of L chains. The kinetics of gene rearrangements were compatible with the classical scheme H----kappa----lambda. A rapid expansion of the expressed repertoire occurred between the weeks 8 and 11, with 95% of the EBV clones having the characteristics of mature B cells. V gene family usage was analyzed for the three loci and compared with the pattern of expression observed at 30 weeks of gestation and in adult clones. The "adult" pattern was rapidly acquired for the H and kappa loci, with the major subgroups being VH3 and V kappa 1. When the expression of the repertoire was "normalized," the pattern of V usage correlated fairly well with the estimated number of VH genes, but differed noticeably for the kappa chains, suggesting that the VH and V kappa repertoires are not regulated by similar processes.

Molecular interactions in the "GAT" idiotypic network: an approach using synthetic peptides.

In order to approach some of the dominant epitopes which are recognized in the GAT (Glu60 Ala30Tyr10)n random terpolymer, a variety of peptides containing 7 to 14 residues were synthesized using glutamic acid and tyrosine as building blocks, and thus were able to mimic determinants common to GAT and GT (Glu50Tyr50)n. One decapeptide and on dodecapeptide were found to inhibit GT-mAb1 (or mAb1') binding to the same extent as GAT. Antibodies were also raised against synthetic peptides which reproduced the sequence of the 6 CDR of the germline anti-GAT Ab1 antibody. Antibodies were obtained against all peptides except L1, and were shown to recognize the native Ab1-Fab. Surprisingly, some of these antibodies also recognized GAT, i.e. anti-L2, anti-H2 and anti-H3, an observation which speaks in favour of a triggering of the idiotypic network at the Ab3 level. Finally, a monoclonal antibody derived from an immunization with an Ab2-D region synthetic peptide was found to be of the Ag+Id- type. Sequence data indicate that the light chain at least is completely different from that the of Ab1/Ab1', which uses only a very precise pair of V germline genes.

A structural basis for the internal image in the idiotypic network: antibodies against synthetic Ab2-D regions cross-react with the original antigen.

In the idiotypic cascade initiated by the random terpolymer (Glu60Ala30Tyr10)n or "GAT", we have identified, in the D region of Ab2 antibodies, either Glu-Glu-Tyr or Tyr-Tyr-Glu sequences which mimic GAT immunodominant epitopes, thus suggesting a structural basis for the internal image. Peptides containing the two D-region characteristic sequences were then synthesized and coupled to BSA. In mice, they elicited antibodies, a fraction of which recognized GAT. These observations speak in favour of the localization of an internal image of the GAT antigen in the D region of Ab2 antibodies.

Preferential expression of VK21E light chains on IdX Ia.7 positive monoclonal anti-I-E antibodies.

We previously characterized major (IdX Ia.7) and minor (IdI) idiotopes in a collection of monoclonal alloantibodies reactive with monomorphic (i.e., Ia.7-like) determinants in the structural domain I of the murine class II I-E molecules. In this report, preliminary structural characterization of this antibody family is presented. First, the contribution of isolated H and L chains of the anti-Ia.7 cluster I mAb 41.A to IdX Ia.7 and IdI 41.A idiotope expression was evaluated by testing the capacity of these chains, either isolated or reassociated in homologous or heterologous hybrid Ig, to inhibit the binding of rat or mouse anti-idiotope mAb to IdX Ia.7+ mAb coated plates. It was found that the IdI 41.A idiotope defined by the mouse anti-idiotopic mAb H90-21.1 required the presence of both 41.A H and L chains for complete expression, while the rat mAb-defined IdX Ia.7 idiotope could be detected on isolated and on reassociated 41.A L chain. To evaluate further the structural correlates of the IdX Ia.7 idiotope, H, L, or both H and L chains of 5 A.BY, 4 A.TH and 1 C3H.SW IdX+ anti-Ia.7 mAb, as well as that of 3 A.TH IdX- anti-I-E or anti-I-A and -I-E mAb were subjected to NH2-terminal amino acid sequencing. These analyses demonstrated a) that different H chains corresponding to different subgroups (at least to the VHII and VHIII) could be expressed without apparent modification of IdX Ia.7 idiotope expression and b) that 9 of 11 IdX+ anti-Ia.7 mAb utilized highly homologous L chains of the VK21E subgroup. The relevance of these findings to the genetic control of the idiotypic markers identified in the Ia.7 system is discussed.

V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.