| 12237220 |
Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis. |
10.1016/s0162-0134(02)00479-8 |
J. Inorg. Biochem. |
Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis.
Abstract
- The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.
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| 12239124 |
SOM230: a new somatostatin peptidomimetic with potent inhibitory effects on the growth hormone/insulin-like growth factor-I axis in rats, primates, and dogs |
10.1210/en.2002-220219. |
Endocrinology |
SOM230: a new somatostatin peptidomimetic with potent inhibitory effects on the growth hormone/insulin-like growth factor-I axis in rats, primates, and dogs
Abstract
- The goal of this project was to find a somatostatin (SRIF) analog with superior therapeutic potential. Receptor binding studies of new SRIF analogs were used to reveal SRIF substructures that interact with individual human SRIF receptor subtypes (sst1-sst5). Incorporation of these substructures into a stable cyclohexapeptide template led to SOM230, which binds with nanomolar affinity to sst1, sst2, sst3, and sst5. In rats, the inhibitory effect of SOM230 on GH was similar to SMS 201-995 (octreotide) at 1 h, but was 4-fold more potent at 6 h post injection, indicating increased metabolic stability. Treatment of rats with SOM230, at 1 and 10 micro g/kg.h, decreased IGF-I plasma levels, on d 2, by 68% and 90% (P < 0.01); whereas, under SMS 201-995 treatment, plasma IGF-I levels decreased by 28% and 49%, respectively. After a 2-wk infusion of rats, the suppression of IGF-I levels by SOM230 was still pronounced, whereas the response to SMS 201-995 was largely lost. This enhanced effect of SOM230 on IGF-I plasma levels was confirmed in an 8-wk study where both analogs were infused at 50 micro g/kg/h in rats. In rhesus monkey, SOM230 and SMS 201-995 treatment resulted in GH inhibition, with half-maximal inhibitory dose values of 0.5 and 0.4 micro g/kg, respectively, but plasma IGF-I levels were only lowered by SOM230 (-53%). In cynomolgus monkeys, a 2-wk infusion of SOM230, but to a much lesser extent of SMS 201-995, lowered plasma GH levels significantly (from 16.3 to 1.8 ng/ml, P = 0.007). Both in cynomolgus monkeys and beagle dogs, infusion of SOM230, but not SMS 201-995, lowered IGF-I levels significantly. In conclusion, SOM230 has a unique structure, binds almost universally to human ssts, and inhibits potently the GH/IGF-I axis cross-species. SOM230 is a candidate drug for clinical use.
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| 12242595 |
Diversity of the penaeidin antimicrobial peptides in two shrimp species |
10.1007/s00251-002-0487-z. |
Immunogenetics |
Diversity of the penaeidin antimicrobial peptides in two shrimp species
Abstract
- Penaeidins, a unique family of antimicrobial peptides (AMPs) with both proline and cysteine-rich domains, were initially identified in the hemolymph of the Pacific white shrimp, Litopenaeus vannamei. Described here are the results of an investigation of penaeidin diversity in individual shrimp from two species, L. vannamei and L. setiferus (Atlantic white shrimp). We report the discovery of a novel penaeidin class, designated penaeidin 4 present in both L. vannamei and L. setiferus, and that all penaeidin classes were expressed in a single individual. In addition, nearly all penaeidins, regardless of class, shared an identical leader sequence while differing dramatically in the remainder of the peptide. Several new class 3 isoforms were identified, as well as sequence variants of Lv3a, which differ in the 3' untranslated region. Penaeidin sequence variability (especially of class 3), within and between individuals, is not interpretable as simple allelic polymorphism and may reflect alternate transcriptional mechanisms. Penaeidins are encoded by a small number of genetic loci and are not likely representatives of a large gene family produced by whole gene duplication, but rather may be products of a multi-component locus. Based on phylogenetic analysis, penaeidins fall into three classes where 1 and 2 are combined while classes 3 and 4 remain distinct. Phylogenetic analysis indicates that all classes of penaeidin were likely present in both species prior to speciation.
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| 12270180 |
Successful bridging from a peptide to a non peptide antagonist at the human tachykinin NK-2 receptor |
10.1016/s0960-894x(02)00539-5. |
Bioorg Med Chem Lett |
Successful bridging from a peptide to a non peptide antagonist at the human tachykinin NK-2 receptor
Abstract
- Non peptide products have been found to show nanomolar binding and functional affinities at the human tachykinin NK-2 receptor. The new antagonists do not possess stereogenic centers and their thermal behaviour in solution is featured by a peculiar set of conformational stereoisomers. A macroscopic viewpoint is preferentially adopted to rationalize the obtained results.
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| 12297050 |
Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains |
10.1016/s0092-8674(02)00963-7. |
Cell |
Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains
Abstract
- Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 A resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique "receptor-mediated dimerization" was verified by EGFR mutagenesis.
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| 12323074 |
The cyclic dipeptide CI-4 [cyclo-(l-Arg-d-Pro)] inhibits family 18 chitinases by structural mimicry of a reaction intermediate |
10.1042/BJ20021034. |
Biochem J |
The cyclic dipeptide CI-4 [cyclo-(l-Arg-d-Pro)] inhibits family 18 chitinases by structural mimicry of a reaction intermediate
Abstract
- Family 18 chitinases are attractive targets for the development of new inhibitors with chemotherapeutic potential against fungi, insects and protozoan/nematodal parasites. Although several inhibitors have been identified, these are based on complex chemistry, which hampers iterative structure-based optimization. Here we report the details of chitinase inhibition by the natural product peptide CI-4 [ cyclo -(L-Arg-D-Pro)], which possesses activity against the human pathogenic fungus Candida albicans, and describe a 1.7 A (0.17 nm) crystal structure of CI-4 in complex with the enzyme. The structure reveals that the cyclic dipeptide inhibits chitinases by structurally mimicking a reaction intermediate, and could, on the basis of its accessible chemistry, be a candidate for further optimization.
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| 12350163 |
Hirsutellide A, a new antimycobacterial cyclohexadepsipeptide from the entomopathogenic fungus Hirsutella kobayasii |
10.1021/np020055+. |
J Nat Prod |
Hirsutellide A, a new antimycobacterial cyclohexadepsipeptide from the entomopathogenic fungus Hirsutella kobayasii
Abstract
- A new cyclohexadepsipeptide, named hirsutellide A (1), was isolated from a cell extract of the entomopathogenic fungus Hirsutella kobayasii BCC 1660. The structure of 1 was elucidated by analyses of spectroscopic data, and its absolute stereochemistry was addressed by the use of Marfey's method. Hirsutellide A (1) exhibited antimycobacterial and antimalarial activities, but was inactive toward the Vero cell line (at 50 microg/mL).
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| 12353082 |
Two new beta3 integrin mutations in Indian patients with Glanzmann thrombasthenia: localization of mutations affecting cysteine residues in integrin beta3 |
10.1055/s-0037-1613244. |
Thromb Haemost |
Two new beta3 integrin mutations in Indian patients with Glanzmann thrombasthenia: localization of mutations affecting cysteine residues in integrin beta3
Abstract
- New mutations in the beta3 integrin subunit have been identified in two unrelated Glanzmann thrombasthenia patients originating from India and Bangladesh. Both patients had histories of excessive bleeding and were found to have Glanzmann thrombasthenia based on absent ADP-induced platelet aggregation. Immunoblotting of platelet lysates of Patient 1 demonstrated reduced levels of alphaIIb and an unexpected high Mr beta3 band of approximately 260,000, with little or no normal-sized beta3. Upon reduction, a weak beta3 band of normal Mr was observed. Platelet lysates of Patient 2 demonstrated undetectable levels of beta3. Sequence analyses identified homozygous mutations in the beta3 genes of both patients. Patient 1 had a C506Y missense mutation resulting in the expression of an unpaired cysteine; we propose that the Mr approximately 260,000 band is a disulfide-bonded beta3 dimer. Patient 2 had an insertion mutation resulting in a frameshift and premature termination. Both mutations affect biogenesis of platelet alphaIIbbeta3 receptors.
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| 12354939 |
Histone deacetylase 6 binds polyubiquitin through its zinc finger (PAZ domain) and copurifies with deubiquitinating enzymes. |
10.1073/pnas.172511699 |
Proc. Natl. Acad. Sci. U.S.A. |
Histone deacetylase 6 binds polyubiquitin through its zinc finger (PAZ domain) and copurifies with deubiquitinating enzymes.
Abstract
- Histone deacetylases (HDACs) are thought to function as critical mediators of transcriptional repression. However, the physiological targets and posttranslational modifications of the class II HDACs are largely unknown. Here we show that the C terminus of HDAC 6 is both necessary and sufficient for specific association with polyubiquitin. This region contains a putative zinc finger but lacks significant similarity to other known ubiquitin binding domains. Thus, we have designated this region as a PAZ domain, for Polyubiquitin Associated Zinc finger. Although the PAZ domain possesses homology with the zinc finger of deubiquitinating enzymes, it is dispensable for the deubiquitinating activity we find associated with HDAC6 following immunopurification. We also show that both HDAC 5 and HDAC 6 are ubiquitinated in vitro and in vivo. However, both of these proteins are stable in vivo and do not appear to be targeted for rapid degradation by the proteasome. Thus, HDAC6 is linked to the ubiquitin system via ubiquitin conjugation, polyubiquitin binding, and copurification with deubiquitinating enzymes.
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| 12355496 |
Antiarthritic activity of an orally active C5a receptor antagonist against antigen-induced monarticular arthritis in the rat |
10.1002/art.10449. |
Arthritis Rheum |
Antiarthritic activity of an orally active C5a receptor antagonist against antigen-induced monarticular arthritis in the rat
Abstract
- To determine if the new, orally active C5a receptor antagonist, the cyclic peptide AcF-[OPdChaWR], reduces the severity of pathology in a rat model of immune-mediated monarticular arthritis.
Arthritis was induced in the right knee of previously sensitized rats by the intraarticular injection of methylated bovine serum albumin. Rats were examined for either 14 days or 28 days, or for 49 days following a second antigen challenge at 28 days. The C5a antagonist (1 or 3 mg/kg/day) and/or ibuprofen (30 mg/kg/day) were administered orally on a daily basis either before or after arthritis induction.
Rats receiving AcF-[OPdChaWR] had significant reductions in right knee swelling, gait disturbance, lavaged joint cell numbers, and right knee histopathology, as well as in serum levels of tumor necrosis factor alpha (TNFalpha) and intraarticular levels of interleukin-6 and TNFalpha on day 14. In the 14- and 28-day studies, ibuprofen resulted in a similar reduction in gait abnormalities and intraarticular inflammatory cells compared with the C5a antagonist, but was less effective in reducing knee swelling over the course of the study and had no effect on knee histopathology. Combination therapy with AcF-[OPdChaWR] and ibuprofen resulted in no greater efficacy than with the C5a antagonist alone. Rats injected twice with the antigen in the 49-day study displayed the most severe histopathology and this, as well as knee swelling and gait abnormalities, was significantly reduced by repeated treatment with the C5a antagonist.
An agent that inhibits the action of C5a in this model significantly reduced joint pathology, while ibuprofen was not effective. C5a antagonists could therefore have broader therapeutic benefits than nonsteroidal antiinflammatory drugs as antiarthritic agents for rheumatoid arthritis.
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| 12357034 |
Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophilin. |
10.1073/pnas.212504399 |
Proc. Natl. Acad. Sci. U.S.A. |
Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophilin.
Abstract
- Calcineurin (Cn), a Ca(2+)/calmodulin-dependent Ser/Thr protein phosphatase, is an important participant in signaling pathways that activate T cells. It is the target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. These drugs bind proteins known as cyclophilin (Cyp) and FK506-binding protein, respectively, and the drug-protein complexes in turn inhibit Cn. We report the crystal structure of a Cyp/CsA/Cn ternary complex, determined to a resolution of 3.1 A. Residues 3-9 of CsA, particularly N-methyl leucines 4 and 6, and Trp-121 of Cyp form a composite surface for interaction with Cn. The hydrophobic interface buries two hydrogen bonds. The structure accounts clearly for the effects of mutations in Cn on CsA-resistance and for the way modifications of CsA alter immunosuppressive activity.
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| 12359626 |
Pharmacological characterization of SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide), a novel peptidic urotensin-II receptor antagonist |
10.1038/sj.bjp.0704887. |
Br J Pharmacol |
Pharmacological characterization of SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide), a novel peptidic urotensin-II receptor antagonist
Abstract
- 1. Human urotensin-II (hU-II), a cyclic undecapeptide, is amongst the most potent mammalian vasoconstrictors identified, suggesting that hU-II and its G-protein-coupled receptor (UT) may regulate cardiovascular homeostasis. Such a hypothesis would benefit greatly from the development of selective UT antagonists. 2. Although the somatostatin (SST) antagonist SB-710411 (Cpa-c[D-Cys-Pal-D-Trp-Lys-Val-Cys]-Cpa-amide) is purported to block U-II-induced contractions in rat isolated aorta, little is known about its specific pharmacological properties. 3. SB-710411 (10 micro M) inhibited hU-II-induced contraction in rat isolated aorta causing a significant, parallel shift in the agonist concentration-response curve (pK(b) 6.28+/-0.11; n=8) with no suppression of the E(max). In contrast, SB-710411 did not alter the contractile actions of angiotensin-II, phenylephrine, or KCl. Paradoxically, however, SB-710411 potentiated the contractile response to endothelin-1 (pEC(50) 8.02+/-0.16 and 8.54+/-0.11, P<0.01; n=8). Rather than being specific toSB-710411, this phenomenon appears to be related to somatostatin receptor affinity and not intrinsic activity since the SST agonist somatostatin-14 and antagonist cyclo-somatostatin also potentiated endothelin-1-induced contraction. 4. SB-710411 (10 micro M) did not inhibit carbachol, sodium nitroprusside, IBMX, isoprenaline, and levcromakalim-induced reversal of tone established with noradrenaline. In contrast, however, SB-710411 significantly inhibited the reversal of tone established with endothelin-1 using the same vasorelaxants. 5. In summary, although SB-710411 inhibits the vasoconstrictor actions of hU-II in a competitive, surmountable manner, it also possesses additional pharmacological actions. Thus, whilst the present study is amongst the first to detail the properties of a functional U-II receptor antagonist, the data suggest caution be used when assessing data generated utilizing this moiety and other SST analogues.
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| 12359715 |
Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling |
10.1074/jbc.M208946200. |
J Biol Chem |
Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling
Abstract
- Membrane-associated adaptors play an important role in coupling antigen receptor engagement to downstream signaling events, such as Ras-MAPK activation, Ca(2+) flux, and nuclear factor of activated T cells (NFAT) activation. Here we identified a novel membrane-associated adaptor protein, LAX. LAX is mainly expressed in B cells, T cells, and other lymphoid-specific cell types. It shares no overall sequence homology with LAT and is not localized to lipid rafts. However, like LAT, LAX has tyrosine motifs for binding Grb2, Gads, and the p85 subunit of phosphatidylinositol 3-kinase. Upon stimulation via the B or T cell receptors, LAX is rapidly phosphorylated by Src and Syk family tyrosine kinases and interacts with Grb2, Gads, and p85. Overexpression of LAX in Jurkat cells specifically inhibits T cell receptor-mediated p38 MAPK activation and NFAT/AP-1 transcriptional activation. Our data suggested that LAX functions to negatively regulate signaling in lymphocytes.
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| 12369825 |
Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives |
10.1021/bi026185z. |
Biochemistry |
Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives
Abstract
- Tachyplesin I is a 17-residue peptide isolated from the horseshoe crab, Tachypleus tridentatus. It has high antimicrobial activity and adopts a beta-hairpin conformation in solution stabilized by two cross-strand disulfide bonds. We report an NMR structural investigation of wild-type tachyplesin I and three linear derivatives (denoted TPY4, TPF4, and TPA4 in which the bridging cysteine residues are uniformly replaced with tyrosine, phenylalanine, and alanine, respectively). The three-dimensional aqueous solution structures of the wild type and the active variant TPY4 reveal very similar beta-hairpin conformations. In contrast, the inactive variant TPA4 is unstructured in solution. The arrangement of the tyrosine side chains in the TPY4 structure suggests that the beta-hairpin is stabilized by aromatic ring stacking interactions. This is supported by experiments in which the beta-hairpin structure of TPF4 is disrupted by the addition of phenol, but not by the addition of an equimolar amount of cyclohexanol. We have also determined the structures of wild-type tachyplesin I and TPY4 in the presence of dodecylphosphocholine micelles. Both peptides undergo significant conformational rearrangement upon micelle association. Analysis of the micelle-associated peptide structures shows an increased level of exposure of specific hydrophobic side chains and an increased hydrophobic integy moment. Comparison of the structures in micelle and aqueous solution for both wild-type tachyplesin I and TPY4 reveals two requirements for high antimicrobial activity: a beta-hairpin fold in solution and the ability to rearrange critical side chain residues upon membrane association.
|
| 12384590 |
Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157 |
10.1093/nar/gkf566. |
Nucleic Acids Res |
Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157
Abstract
- We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.
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