Copper(I) Complexes of N-(2-{[(2E)-2-(4-Nitrobenzylidenyl)Hydrazinyl]Carbonyl}Phenyl)Benzamide and Triphenylphosphine: Synthesis, Characterization and Luminescence Properties.

Copper(I) complexes of the formula [Cu(L)(PPh3)2]X (1-4) (X = Cl(1), ClO4(2), BF4(3) and PF6(4)) [where L = N-(2-{[(2E)-2-(4-nitrobenzylidenyl)hydrazinyl]carbonyl}phenyl)benzamide; PPh3 = triphenylphosphine] have been prepared by the condensation of N-[2-(hydrazinocarbonyl)phenyl]benzamide with 4-nitrobenzaldehyde followed by the reaction with CuCl, [Cu(MeCN)4]ClO4, [Cu(MeCN)4]BF4 and [Cu(MeCN)4]PF6 in presence of triphenylphosphine as a coligand. Complexes 1-4 were then characterized by elemental analyses, FTIR, UV-visible and 1H NMR spectroscopy. Mononuclear copper(I) complexes 1-4 were formed with L in its keto form by involvement of azomethine nitrogen and the carbonyl oxygen along with two PPh3 groups. A single crystal X-ray diffraction study of the representative complex [(Cu(L)(PPh3)2]CIO4 (2) reveals a distorted tetrahedral geometry around Cu(I). Crystal data of (2): space group = C2/c, a = 42.8596 (9) Å, b = 14.6207 (3) Å, c = 36.4643 (7) Å, V = 20,653.7 (7) Å3, Z = 16. Complexes 1-4 exhibit quasireversible redox behaviour corresponding to a Cu(I)/Cu(II) couple. All complexes show blue-green emission as a result of fluorescence from an intra-ligand charge transition (ILCT), ligand to ligand charge transfer transition (LLCT) or mixture of both. Significant increase in size of the counter anion shows marked effect on quantum efficiency and lifetime of the complexes in solution.

The primary structure of the human leukocyte antigen CD37, a species homologue of the rat MRC OX-44 antigen.

Comparison of NH2-terminal protein sequence from the rat OX-44 antigen with the sequence of the human CD37 antigen deduced from a cDNA clone shows that these antigens are species homologues. The CD37 sequence is 244 amino acids in length and lacks a conventional leader sequence. The molecule is likely to have an NH2-terminal cytoplasmic domain followed by three transmembrane sequences that lie within the first 110 amino acids. The rest of the molecule is hydrophillic and contains three sites for N-linked glycosylation.

Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q.

This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular "heads" of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH2-terminal amino acid sequence of the first 24 residues of the C1q-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue-long NH2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of approximately 60 kD. The NH2-terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

Similarities in sequences and cellular expression between rat CD2 and CD4 antigens.

The MRC OX-34 antigen of rat T lymphocytes was purified and peptide sequences were obtained. Oligonucleotide probes were synthesized and cDNA clones coding for the antigen were isolated and sequenced to yield a predicted protein sequence for the molecule that fitted the peptide data. Comparison of this sequence with that for human CD2 determined by Sewell et al. showed that OX-34 is rat CD2. The primary structure of the molecule was notable for a moderately large cytoplasmic domain of unusual sequence and also for its highly significant relationship to CD4 antigen in the membrane proximal extracellular region and the transmembrane sequence. A relationship to the Ig superfamily can be argued for the two extra cellular domains of CD2, even though neither fits the standard pattern for Ig-related domains. Within the T lymphocyte lineage, rat CD2 seemed to be present on all stages with the exception of approximately 50% of the thymic CD4-,CD8- cells. In addition, the antigen was prominent on most macrophages in the spleen but not found on peritoneal or liver macrophages. CD4 antigen is also expressed on T lymphocytes and macrophages, and thus CD2 and CD4 appear similar in their cellular expression as well as structural characteristics.

HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides.

Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

Porcine natural-killer-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro.

Natural-killer-enhancing factor-B (NKEF-B) (monomeric mass = 21.82 kDa) was purified from the cytosol of porcine red blood cells and its identity was established by microsequencing. NKEF-B oligomerisation was investigated by gel filtration and small-angle X-ray scattering (SAXS). Native NKEF-B readily forms disulphide-linked dimers, but when fully reduced, the protein forms discrete oligomers containing 16 +/- 1 monomers. A total of 40% of the purified enzyme was deduced to be cysteinylated, which is consistent with the modification of one or both of two putative active site cysteine residues. In vitro, NKEF-B was found to be a specific substrate of mu- and m-calpains, the calcium-dependent cysteine proteases. The cleavage events were followed by SDS-PAGE and the cleavage sites pinpointed by N-terminally sequencing the resulting digestion fragments. This in vitro cleavage data provides support to the hypothesis that calpromotin (NKEF-B), an erythron peroxiredoxin involved in the regulation of calcium-dependent potassium transport across the plasma membrane, is cleaved by calpain in vivo.

Primary structure and properties of the inhibitory protein of the mitochondrial ATPase (H(+)-ATP synthase) from potato.

The primary structure of the inhibitory protein (IF1) of the potato mitochondrial ATPase has been determined by protein sequencing, and its molecular weight determined by electrospray mass spectrometry. Both are consistent with a 56-residue protein of molecular weight 6697. This protein shows only weak homology with IF1 sequences from mammals and yeasts, and significant deletions are present compared to these sequences. Homology is strongest in the region between residues 22 and 46 (ox heart numbering), where 5 identities and 6 conserved residues are observed across all five IF1 species. In addition, this region shows homology with protein inhibitors from ATPases other than mitochondrial F1. It is suggested that this region might constitute an ATPase 'inhibitory motif'. Functional studies show that, unlike IF1 from mammals or yeasts, potato IF1 binds only poorly to ox heart F1, and does not show the ability to exist in 2 (alternate) stable conformations.

Amylin and the amylin gene: structure, function and relationship to islet amyloid and to diabetes mellitus.

Amylin, the major peptide component of the islet amyloid commonly found in the pancreases of patients with type 2 (non-insulin-dependent) diabetes mellitus (NIDDM), is a recently discovered islet polypeptide. This peptide has many structural and functional features suggesting that it is a novel hormone, which may control carbohydrate metabolism in partnership with insulin and other glucoregulatory factors. Amylin is synthesised in, and probably secreted from, the beta-cells of the islets of Langerhans, where it has recently been immunolocalised to secretory granules. DNA cloning studies indicate that in the human and the rat, amylin is generated from a precursor, preproamylin, which displays a typical signal peptide followed by a small prohormone-like sequence containing the amylin sequence. The presence of the signal peptide suggests that amylin is secreted and plays a physiological role. Amylin is probably generated by proteolytic processing similar to that for proinsulin and other islet prohormones. The human amylin gene encodes the complete polypeptide precursor in two exons which are separated by an intron of approx. 5 kb, and is located on chromosome 12. Amylin is a potent modulator of glycogen synthesis and glucose uptake in skeletal muscle, and is capable of inducing an insulin-resistant state in this tissue in vitro, and perhaps also in the liver in vivo. In normal metabolism, amylin could act in concert with insulin as a signal for the body to switch the site of carbohydrate disposal from glycogen to longer-term stores in adipose tissue, by making skeletal muscle relatively insulin-resistant, whilst at the same time leaving rates of insulin-stimulated carbohydrate metabolism in adipose tissue unaltered. Several lines of evidence now implicate elevated amylin levels in the pathogenic mechanisms underlying NIDDM, and suggest to us that the obesity which frequently accompanies this syndrome is a result of, rather than a risk factor for, NIDDM. Following the beta-cell destruction which occurs in type 1 (insulin-dependent) diabetes mellitus (IDDM), it is probable that amylin secretion disappears in addition to that of insulin. As patients with insulin-treated IDDM frequently experience problems with hypoglycaemia, and as amylin acts to modulate the action of insulin in various tissues, it is possible that amylin deficiency may contribute to morbidity in insulin-treated IDDM, perhaps through the loss of a natural damping mechanism which guards against hypoglycaemia under conditions of normal physiology.

Cytochrome cd1 structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to beta-propeller folds.

The central tunnel of the eight-bladed beta-propeller domain of cytochrome cd1 (nitrite reductase) is seen, from a 1.28 A resolution structure, to contain hydrogen donors and acceptors that are satisfied by interaction either with water or the d1 haem. The d1 haem, although bound by an extensive network of hydrogen bonds, is not distorted in its binding pocket and is confirmed to have exactly the dioxoisobacteriochlorin structure proposed from chemical studies. A biological rationale is advanced for the undistorted structure of the d1 haem and the large number of hydrogen bonds it makes. The beta-propeller domain can be closely superimposed on that of methanol dehydrogenase despite the enzymes sharing no common sequence motifs and using a different set of interactions to "Velcro" close the propeller. The sequence and likely structural relationships between cytochrome cd1 or methanol dehydrogenase and other predicted eight-bladed beta-propeller domains in proteins, such as the pyrolloquinoline quinone-dependent alcohol dehydrogenase, are discussed and compared with other propeller proteins. From sequencing the nirS gene of Thiosphaera pantotropha, it is established that the amino acid sequence deduced previously in part from X-ray diffraction data at lower resolution was largely correct, as was the proposal that eight N-terminal amino acid residues were not seen in the structure. The unusual haem iron environments in both the c-type cytochrome domain, with His/His coordination, and the d1-type cytochrome domain with Tyr/His coordination are related to the functions of the redox centres.

Isolation of a human endothelial cell C1q receptor (C1qR).

C1q binding to endothelial cells has been described previously, but the putative cell surface receptor(s) has not been identified. In the present study, modifications of a reported purification of lymphocyte C1q receptor (C1qR) were used to isolate C1q binding sites from human umbilical vein endothelial cells. Cells were harvested, without protease treatment, at passage 10-17 and lysed with 1% Triton X-100. The lysate was fractionated on Fast-performance liquid chromatography (FPLC) Mono-Q using a linear NaCl gradient, followed by high-performance liquid chromatography (HPLC) ion exchange (TSKgel DEAE-NPR). A major protein was eluted that had the same mobility on sodium dodecyl sulfate-polyacrylamide gels and the same NH2-terminal sequence as lymphocyte C1qR. This protein was expressed on the surface, as judged by surface radioiodination, bound to C1q-coated surfaces, and was recognized by polyclonal antilymphocyte C1qR antibodies. Thus, endothelial cells express a C1q receptor that appears identical to lymphocyte C1qR. The data further support the hypothesis that cell surface C1qRs identified on a variety of somatic and cultured cells are either identical or constitute a family of closely related molecules.

Identification of the CD85 antigen as ILT2, an inhibitory MHC class I receptor of the immunoglobulin superfamily.

The CD85 molecule was originally defined at the Fifth Workshop on Leucocyte Antigens in 1993 by two monoclonal antibodies, VMP55 and GHI/75. This cell-surface glycoprotein is expressed on B cells, monocytes, and subpopulations of T and natural killer (NK) cells, and particularly high levels are expressed by normal and neoplastic plasma cells and by hairy cell leukemia B cells. We affinity purified the CD85 antigen and obtained tryptic peptide sequence which indicated that this molecule might be ILT2, a recently described inhibitory major histocompatibility complex class I receptor of the immunoglobulin superfamily. This was confirmed by showing that both of the original anti-CD85 mAbs stained ILT2 transfectants. The cell signaling role demonstrated for ILT2 is consistent with the previously reported involvement of CD85 in T cell activation.

The promise and obstacle of p53 as a cancer therapeutic agent.

p53 is a tumor suppressor gene that is mutated in greater than 50% of human cancers. The action of p53 as a tumor suppressor involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Loss of p53 function therefore allows the uncontrolled proliferation associated with cancerous cells. While design of most anti-cancer agents has focused on targeting and inactivating cancer promoting targets, such as oncogenes, recent attention has been given to restoring the lost activity of tumor suppressor genes. Because the loss of p53 function is so prevalent in human cancer, this protein is an ideal candidate for such therapy. Several gene therapeutic strategies have been employed in the attempt to restore p53 function to cancerous cells. These approaches include introduction of wild-type p53 into cells with mutant p53; the use of small molecules to stabilize mutant p53 in a wild-type, active conformation; and the introduction of agents to prevent degradation of p53 by proteins that normally target it. In addition, because mutant p53 has oncogenic gain of function activity, several approaches have been investigated to selectively target and kill cells harboring mutant p53. These include the introduction of mutant viruses that cause cell death only in cells with mutant p53 and the introduction of a gene that, in the absence of functional p53, produces a toxic product. Many obstacles remain to optimize these strategies for use in humans, but, despite these, restoration of p53 function is a promising anti-cancer therapeutic approach.

MRC OX-45 antigen: a leucocyte/endothelium rat membrane glycoprotein of 45,000 molecular weight.

Monoclonal antibodies MRC OX-45 and OX-46 detect identical or juxtaposed antigenic determinants on a novel rat membrane molecule that plays a possible role in macrophage suppression of antigen-induced T-cell responses. These antibodies react with most mature hematopoietic cells and their bone-marrow precursors, vascular endothelium and some connective tissue. The OX-45 antigens were purified from brain (mainly endothelium) and spleen by immunoaffinity chromatography, and were found to be glycoproteins with apparent Mr 43,000 and 45,000, respectively, as determined by SDS-PAGE analysis. The amino acid compositions of the two preparations were very similar but with no distinguishing features. The broad pattern of distribution was not the result of fortuitous cross-reaction of the MAbs as a single N-terminal sequence was obtained from mixed spleen populations of cells. Carbohydrate compositions of the brain and spleen molecules differed both in absolute amount (22 and 41% by weight, respectively) and in the ratios of various saccharides reflecting overall differences in the patterns of glycosylation between the two tissues. MRC OX-45 IgG showed an heterogeneity in the Mr of its H chain due to the attachment, in some molecules, of carbohydrate structures to the Fd fragment.

Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation.

The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface-bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site-directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B-like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A-like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group-containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s-1 (t1/2 of 10.3 s) for C4A and 1.08 s-1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.

Interactions of the C Terminus of Metabotropic Glutamate Receptor Type 1α with Rat Brain Proteins: Evidence for a Direct Interaction with Tubulin.

Metabotropic glutamate receptors (mGluRs) are coupled to G protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. As part of a strategy for defining the mechanisms for the specific targeting of mGluR1 α, rat brain proteins which interact with the intracellular carboxy terminus of mGluR1 α have been characterized, using affinity chromatography on a glutathione S-transferase fusion protein that contains the last 86 amino acids of mGluR1 α. Three of the proteins specifically eluted from the affinity column yielded protein sequences, two of which were identified as glyceraldehyde-3-phosphate dehydrogenase and ß-tubulin ; the other was an unknown protein. The identity of tubulin was confirmed by western immunoblotting. Using a solid-phase binding assay, the mGluR1 α-tubulin interaction was shown to be direct, specific, and saturable with a KD of 2.3 ± 0.4 µM. In addition, mGluR1 α, but not mGluR2/3 or mGluR4, could be coimmunoprecipitated from solubilized brain extracts with tubulin using anti-ß-tubulin antibodies. However, mGluR1 α could not be coimmunoprecipitated with the tubulin binding protein gephyrin, nor could it be coimmunoprecipitated with PSD95. Collectively these data demonstrate that the last 86 amino acids of the carboxyl-terminal tail of mGluR1 α are sufficient to determine its interaction with tubulin and that there is an association of this receptor with tubulin in rat brain.

Biotinyl analogues of amylin as biologically active probes for amylin/CGRP receptor recognition.

Biotinyl analogues of rat amylin were synthesised with sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate (NHS-SS-Biotin). Biotinylated amylin peptides were purified by HPLC, quantitated, and the presence of the biotin group at Lys-1 confirmed by peroxidase-labelled avidin and FAB mass spectroscopy. Amylin-biotin retained a similar affinity for binding to rat liver plasma membranes compared with rat amylin and also completely inhibited insulin-stimulated glycogen synthesis in rat soleus muscle incubated in vitro. These biologically active amylin probes will enable a complete analysis of amylin/CGRP receptor expression in various cell types and facilitate the isolation and characterisation of the hormone-receptor complex.

Human interleukin-1 receptor antagonist. High yield expression in E. coli and examination of cysteine residues.

The human IL-1 receptor antagonist (IL-1ra) was produced in a high yield E. coli expression system, and was purified in a rapid two-step purification. This recombinant IL-1ra molecule possessed full binding activity to the IL-1 receptor (type I) and totally inhibited IL-1-induced PGE2 production by human dermal fibroblasts. Radioalkylation and analysis of V8-derived IL-1ra peptides indicate that the four cysteines present in the IL-1ra are not disulphide-linked.

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.