We cloned 17 different PCR fragments encoding VH genes of camel (Camelus dromedarius). These clones were derived from the camel heavy chain immunoglobulins lacking the light chain counterpart of normal immunoglobulins. Insight into the camel VH sequences and structure may help the development of single domain antibodies. The most remarkable difference in the camel VH, consistent with the absence of the VL interaction, is the substitution of the conserved Leu45 by an Arg or Cys. Another noteworthy substitution is the Leu11 to Ser. This amino acid normally interacts with the CH1 domain, a domain missing in the camel heavy chain immunoglobulins. The nature of these substitutions agrees with the increased solubility behavior of an isolated camel VH domain. The VH domains of the camels are also characterized by a long CDR3, possibly compensating for the absence of the VL contacts with the antigen. The CDR3 lacks the salt bridge between Arg94 and Asp101. However, the frequent occurrence of additional Cys residues in both the CDR1 and CDR3 might lead to the formation of a second internal disulfide bridge, thereby stabilizing the CDR structure as in the DAW antibody. Within CDRs of the camel VH domains we observe a broad size distribution and a different amino acid pattern compared with the mouse or human VH. Therefore the camel hypervariable regions might adopt structures which differ substantially from the known canonical structures, thereby increasing the repertoire of the camel antigen binding sites within a VH.

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p-and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast. INTRODUCTIONThe mitogen-activated protein (MAP) kinase (MAPK) signaling pathways are critical for the response of cells to changes in their environment (Marshall, 1994;Herskowitz, 1995;Waskiewicz and Cooper, 1995;Treisman, 1996). They serve to transduce signals generated at the cell surface or in the cytoplasm to the nucleus, where changes in gene expression result. In mammalian cells, multiple distinct MAP kinases have been identified, including a large subset whose members are activated by a variety of environmental stress conditions, DNA-damaging agents, inflammatory cytokines, and certain vasoactive neuropeptides Freshney et al., 1994;Galcheva-Gargova et al., 1994;Han et al., 1994;Kyriakis et al., 1994;Lee et al., 1994;Rouse et al., 1994;Sluss et al., 1994). These stress-activated MAP kinases (SAPKs) fall into two distinct classes, termed the C-Jun N-terminal kinase ( JNK) and p38 kinases, based on their sequences (Davies, 1994;Waskiewicz and Cooper, 1995). A number of transcription factors are phosphorylated in response to SAPK activation; for example, the c-Jun factor is regulated by JNK (Hibi et al., 1993;Derijard et al., 1994;Kyriakis et al., 1994) but not by p38, whereas ATF2 is phosphorylated and regulated by both JNK Livingstone et al., 1995;van Dam et al., 1995) and p38 (Raingeaud et al., 1995). Although a number of MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKKs) that activate the SAPKs have been identified in mammalian cells, very little is known about how these are regulated by stress stimuli (reviewed in Ichijo, 1999;Tibbles and Woodgett, 1999). This is probably due to the multiplicity of SAPK pathways in mammalian cells and the difficulties of genetic analysis in these organisms.Recently, a single member of the SAPK family, called Sty1p (also known as Spc1p or Phh1p), ...

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Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformationswitching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M 1 muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation. Muscarinic acetylcholine receptors (mAChRs)1 belong to the rhodopsin-like family of 7-transmembrane (7-TM) receptors. Agonists bind to these receptors at the extracellular end. This leads to the binding and activation of a G protein at the intracellular face. These receptors are characterized by the possession of a particular set of evolutionarily conserved amino acids, mostly located in the 7-TM helices, implying that they may share a common mechanism of activation. However, the topography of the binding pockets and the conformational changes related to agonist-induced receptor activation are incompletely understood. In recent studies on the M 1 mAChR, we have identified ligand contact residues in TM 3 (1), 5 (2), and 6 (3) and inferred a strip of residues in TM 3 contributing to the activated state of the M 1 mAChR by using alanine-scanning mutagenesis, or cysteine-scanning mutagenesis. The three-dimensional crystal structure of the ground state of rhodopsin at 2.8 Å (4) has provided a framework for modeling the mAChRs and allowed us to interpret most of the information obtained by mutagenesis studies on the M 1 mAChR.In the initial projection map of rhodopsin (5), TM 4 appeared as an outlier of the helical bundle, with a large lipid-exposed surface and few polar residues. Consequently, its function has received little attention. However, a recent photo-activated chromophore cross-linking study of rhodopsin has shown a flip-over of the ionone ring from the neighborhood of TM 6 to TM 4 during receptor activation, implying that a substantial movement of TM ...

BackgroundTRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs).ResultsWe investigated four patients from two families with infantile-onset cyclical, aseptic febrile episodes with vomiting and diarrhoea, global electrolyte imbalance during these episodes, sideroblastic anaemia, B lymphocyte immunodeficiency, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Other clinical features found in children include sensorineural deafness, cerebellar atrophy, brittle hair, partial villous atrophy and nephrocalcinosis.Whole exome sequencing and bioinformatic filtering were utilised to identify recessive compound heterozygous TRNT1 mutations (missense mutation c.668T>C, p.Ile223Thr and a novel splice mutation c.342+5G>T) segregating with disease in the first family. The second family was found to have a homozygous TRNT1 mutation (c.569G>T), p.Arg190Ile, (previously published).We found normal mitochondrial translation products using passage matched controls and functional perturbation of 3’ CCA addition to mitochondrial tRNAs (tRNACys, tRNALeuUUR and tRNAHis) in fibroblasts from two patients, demonstrating a pathomechanism affecting the CCA addition to mt-tRNAs. Acute management of these patients included transfusion for anaemia, fluid and electrolyte replacement and immunoglobulin therapy. We also describe three-year follow-up findings after treatment by bone marrow transplantation in one patient, with resolution of fever and reversal of the abnormal metabolic profile.ConclusionsOur report highlights that TRNT1 mutations cause a spectrum of disease ranging from a childhood-onset complex disease with manifestations in most organs to an adult-onset isolated retinitis pigmentosa presentation. Systematic review of all TRNT1 cases and mutations reported to date revealed a distinctive phenotypic spectrum and metabolic and other investigative findings, which will facilitate rapid clinical recognition of future cases.

The chemokine receptor CCR1 and its principal ligand, CCL3/MIP-1␣, have been implicated in the pathology of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and asthma. As such, these molecules are the focus of much research with the ultimate aim of developing novel therapies. We have described previously a non-competitive small molecule antagonist of CCR1 (UCB 35625), which we hypothesized interacted with amino acids located within the receptor transmembrane (TM) helices (Sabroe, I., Peck, M. J., Jan Van Keulen, B., Jorritsma, A., Simmons, G., Clapham, P. R., Williams, T. J., and Pease, J. E. (2000) J. Biol. Chem. 275, 25985-25992). Here we describe an approach to identifying the mechanism by which the molecule antagonizes CCR1. Thirty-three point mutants of CCR1 were expressed transiently in L1.2 cells, and the cells were assessed for their capacity to migrate in response to CCL3 in the presence or absence of UCB 35625. Cells expressing the mutant constructs Y41A (TM helix 1, or TM1), Y113A (TM3), and E287A (TM7) were responsive to CCL3 but resistant to the antagonist, consistent with a role for the TM helices in CCR1 interactions with UCB 35625. Subsequent molecular modeling successfully docked the compound with CCR1 and suggests that the antagonist ligates TM1, 2, and 7 of CCR1 and severely impedes access to TM2 and TM3, a region thought to be perturbed by the chemokine amino terminus during the process of receptor activation. Insights into the mechanism of action of these compounds may facilitate the development of more potent antagonists that show promise as future therapeutic agents in the treatment of inflammatory disease.

A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.