| 12052189 |
PF1022A and related cyclodepsipeptides - a novel class of anthelmintics |
10.2174/1568026023393624. |
Curr Top Med Chem |
PF1022A and related cyclodepsipeptides - a novel class of anthelmintics
Abstract
- Parasitic nematodes are a major cause of morbidity and mortality in man and also cause widespread loss of food production by infection of livestock. A milestone in the chemotherapy of nematode infections, especially in animals, was the discovery of the avermectins and milbemycins during the 1970s. Since the discovery of these highly active macrolides, reports of potent new classes of anthelmintics have been scarce. One of the most outstanding recently reported anthelmintics is the cyclooctadepsipeptide PF1022A, the most active member of a novel class of anthelmintic agents. During the past years several total syntheses of PF1022A and manifold structure-activity relationships have been established. Additionally, the biosynthesis of PF1022A has been elucidated and intensive investigations into the mode of action of this novel anthelmintic are underway. Comprehensive studies including cyclodepsipeptides with smaller ring-sizes, such as the enniatins, proved the PF1022 family and related cyclodepsipeptides to be the most promising follow-up candidates for the avermectins and milbemycins, which suffer from increasing nematode resistance.
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| 12059813 |
Distribution and function of FSH receptor genetic variants in normal men |
10.1046/j.1439-0272.2002.00493.x. |
Andrologia |
Distribution and function of FSH receptor genetic variants in normal men
Abstract
- Follicle stimulating hormone (FSH) plays a key role in the maintenance of qualitatively and quantitatively normal spermatogenesis. It controls gamete development through Sertoli cells, via binding to its receptor. The influence and importance of FSH receptor (FSHR) variants on Sertoli cell function is not completely understood and remains to be investigated. In this retrospective study, we explored the impact and action of two distinct FSHR isoforms, Thr307/Asn680 and Ala307/Ser680, in a large group of men. This investigation includes 288 normal healthy men, 86 of whom were proven fathers previously studied, and 202 were newly recruited subjects. The FSHR polymorphism at position 680 was analyzed in the whole group, while position 307 was investigated in 150 subjects, both of them by single-stranded conformation polymorphism (SSCP) gel electrophoresis. The distribution frequency for position 680 was 29% for the Asn/Asn, 52% for the Asn-Ser, 19% for the Ser-Ser variant, and for position 307, 27% for the Thr-Thr, 55% for the Ala-Thr, 18% for the Ala-Ala, respectively. Polymorphism combinations that were different from Thr307/Asn680 - Ala307/Ser680 were found in four subjects. When subjects were grouped according to genotype at position 680, no significant differences between basal FSH, testosterone, inhibin B levels and semen parameters were found. This clinical finding demonstrates that, differently from females, in whom a significant correlation between FSHR polymorphism and basal FSH levels was found, the FSHR genotype has no influence on clinical parameters in males.
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| 12061549 |
Identifying protein kinase inhibitors using an assay based on inhibition of aerial hyphae formation in Streptomyces |
10.7164/antibiotics.55.407. |
J Antibiot (Tokyo) |
Identifying protein kinase inhibitors using an assay based on inhibition of aerial hyphae formation in Streptomyces
Abstract
- We have identified a strain of Streptomyces in which aerial hyphae formation appears to be especially sensitive to inhibition by protein kinase inhibitors. Using this assay, a number of bacterial cultures have been screened and novel inhibitors of eukaryotic protein kinases have been identified. Since M. tuberculosis possesses multiple eukaryotic-like protein kinase genes, we tested the active kinase inhibitors for the inhibition of mycobacterial growth and obtained several potent compounds. This identifies a new biochemical class of antimycobacterial agents.
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| 12062679 |
Study of the stability of polymyxins B(1), E(1) and E(2) in aqueous solution using liquid chromatography and mass spectrometry |
10.1016/s0731-7085(02)00016-x. |
J Pharm Biomed Anal |
Study of the stability of polymyxins B(1), E(1) and E(2) in aqueous solution using liquid chromatography and mass spectrometry
Abstract
- Polymyxins B(1), E(1) (colistin A) and E(2) (colistin B) were subjected to degradation in aqueous solutions of different pH values (1.4, 3.4, 5.4 and 7.4) and at different temperatures (37, 50 and 60 degrees C) in order to investigate the characteristics of decomposition. The progress of decomposition was followed by reversed-phase liquid chromatography on YMC-Pack Pro, C-18 stationary phase. The degradation curves showed (pseudo) first order kinetics. The pH-rate profiles indicate that colistin is more susceptible to degradation in solutions of pH above 5 and is more stable in acidic media. The degradation of polymyxin B(1) was most rapid at pH 7.4. Qualitative analysis of the degradation products by LC/MS reveals that racemization is the major mechanism of degradation in both acidic and neutral media.
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| 12072395 |
Identification of an upstream pituitary-active promoter of human somatostatin receptor subtype 5. |
10.1210/endo.143.7.8883 |
Endocrinology |
Identification of an upstream pituitary-active promoter of human somatostatin receptor subtype 5.
Abstract
- Somatostatin receptor subtype 5 (sst5) has been linked to inhibition of PRL and insulin secretion. We characterized the genomic structure of the human sst5. The transcription start site was located 94 nucleotides upstream of the initiator ATG codon. Sequence analysis of 5'-inverse PCR products revealed the presence of a 6.1-kb intron in the 5'-untranslated region. RT-PCR analysis indicated tissue-specific activation of the newly identified upstream promoter in pituitary, but not in small intestine, lung, or placenta. A -1741 promoter directed significant levels of luciferase expression in GH(4) rat pituitary cells, Skut-1B endometrium cells, and JEG3 chorion carcinoma cells, which was absent in COS-7 monkey kidney cells. A minimal -101 promoter was sufficient to allow tissue-specific expression. Its activity in COS-7 cells was not enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. Analysis of deletion constructs revealed a GC-rich region immediately upstream of the transcription start site, which is necessary for promoter activity. Somatostatin led to a significant inhibition, and forskolin and thyroid hormone to a significant stimulation of pituitary-specific promoter activity. Further mapping suggested a cAMP-responsive element located between -101 and the transcription start site, and thyroid hormone-responsive elements between -1741 and -1269 and between -317 and -101. These studies identified an upstream promoter of the sst5 gene with tissue-specific activity.
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| 12082099 |
Solution structure of peptide toxins that block mechanosensitive ion channels |
10.1074/jbc.M202715200. |
J Biol Chem |
Solution structure of peptide toxins that block mechanosensitive ion channels
Abstract
- Mechanosensitive channels (MSCs) play key roles in sensory processing and have been implicated as primary transducers for a variety of cellular responses ranging from osmosensing to gene expression. This paper presents the first structures of any kind known to interact specifically with MSCs. GsMTx-4 and GsMtx-2 are inhibitor cysteine knot peptides isolated from venom of the tarantula, Grammostola spatulata (Suchyna, T. M., Johnson, J. H., Hamer, K., Leykam, J. F., Gage, D. A., Clemo, H. F., Baumgarten, C. M., and Sachs, F. (2000) J. Gen. Physiol. 115, 583-598). Inhibition of cationic MSCs by the higher affinity GsMtx-4 (K(D) approximately 500 nm) reduced cell size in swollen and hypertrophic heart cells, swelling-activated currents in astrocytes, and stretch-induced arrhythmias in the heart. Despite the relatively low affinity, no cross-reactivity has been found with other channels. Using two-dimensional NMR spectroscopy, we determined the solution structure of GsMTx-4 and a lower affinity (GsMTx-2; K(D) approximately 6 microm) peptide from the same venom. The dominant feature of the two structures is a hydrophobic patch, utilizing most of the aromatic residues and surrounded with charged residues. The spatial arrangement of charged residues that are unique to GsMTx-4 and GsMTx-2 may underlie the selectivity of these peptides.
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| 12083483 |
Glanzmann's thrombasthenia: identification of 19 new mutations in 30 patients |
None |
Thromb Haemost |
Glanzmann's thrombasthenia: identification of 19 new mutations in 30 patients
Abstract
- Glanzmann's thrombasthenia (GT) is a genetically heterogeneous autosomal recessive syndrome associated with a bleeding tendency. To elucidate molecular basis of GT we have screened for mutations 30 GT patients. On the whole, 21 different candidate causal mutations, 17 in the alphaIIb and 4 in the beta3 gene have been found. Only two (alphaIIb Pro145Ala and IVS3(-3)-418del) have been previously reported. Nine mutations (42.9%) were likely to produce truncated proteins, whereas the remaining 12 were missense mutations that affected highly conserved residues in alphaIIb and beta3 genes. Six mutations were found in different patients suggesting a possible founder effect. The wide spectrum of expressivity, ranging from mild to severe also among patients carrying the same mutations, provided evidence for a role of different loci or circumstantial factors. In conclusion, we have identified a spectrum of unreported mutations that may be of value to unravel the role of specific regions of alphaIIb and beta3 genes.
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| 12089024 |
Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota |
10.1128/AEM.68.7.3424-3431.2002. |
Appl Environ Microbiol |
Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota
Abstract
- Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota.
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| 12107746 |
An arginine to cysteine(252) mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events |
10.1007/s00125-002-0798-5. |
Diabetologia |
An arginine to cysteine(252) mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events
Abstract
- Aims/hypothesis:
We examined the properties of a mutant insulin receptor (IR) with an Arg(252) to Cys (IR(R252C)) substitution in the alpha-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans.
Methods:
We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IR(R252C).
Results:
Our investigation showed an impairment in insulin binding to IR(R252C) related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IR(R252C) maturation; an inhibition of IR(R252C)-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IR(R252C) on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IR(R252C) followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IR(R252C) as compared to cells expressing IRwt.
Conclusion/interpretation:
These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.
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| 12121902 |
Tn5406, a new staphylococcal transposon conferring resistance to streptogramin a and related compounds including dalfopristin |
10.1128/AAC.46.8.2337-2343.2002. |
Antimicrob Agents Chemother |
Tn5406, a new staphylococcal transposon conferring resistance to streptogramin a and related compounds including dalfopristin
Abstract
- We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.
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| 12138094 |
Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved. |
10.1074/jbc.m202766200 |
J. Biol. Chem. |
Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved.
Abstract
- The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.
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| 12138114 |
Crystal structure of the Apo, unactivated insulin-like growth factor-1 receptor kinase. Implication for inhibitor specificity |
10.1074/jbc.M205580200. |
J Biol Chem |
Crystal structure of the Apo, unactivated insulin-like growth factor-1 receptor kinase. Implication for inhibitor specificity
Abstract
- The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted beta-sheet flanked by a single helix, and the C-terminal lobe comprises eight alpha-helices and four short beta-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121-1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.
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| 12139452 |
Monocyclic human tachykinin NK-2 receptor antagonists as evolution of a potent bicyclic antagonist: QSAR and site-directed mutagenesis studies |
10.1021/jm011127h. |
J Med Chem |
Monocyclic human tachykinin NK-2 receptor antagonists as evolution of a potent bicyclic antagonist: QSAR and site-directed mutagenesis studies
Abstract
- A new series of monocyclic pseudopeptidic tachykinin NK-2 receptor antagonists has been derived from nepadutant with the help of site-directed mutagenesis studies and QSAR models. MEN11558 is the lead compound which is evaluated on a series of 13 new human tachykinin NK-2 receptor mutants (Tyr107Ala, Gln109Ala, Asn110Ala, Phe112Ala, Ser164Phe, Cys167Gly, Phe168Ala, Tyr169Ala, Ile202Phe, Trp263Ala, Tyr269Phe, Tyr269Ala, and Phe293Ala) and 8 mutants on which data from nepadutant were already available (Gln166Ala, Ser170Ala, Thr171Ala, His198Ala, Tyr206Phe, Tyr266Phe, Tyr289Phe, and Tyr289Thr). The results show that the two compounds share most of their binding sites, in agreement with their hypothesized binding modes. This allows us to transfer the structural knowledge we already had for nepadutant to the new series of compounds. At the same time, a sound QSAR model is developed to assist the prioritization of new chemical syntheses. The result is the discovery of receptor antagonists with a higher affinity than nepadutant for the hNK-2 receptor.
|
| 12140263 |
Daxx and histone deacetylase II associate with chromatin through an interaction with core histones and the chromatin-associated protein Dek. |
10.1242/jcs.115.16.3319 |
J. Cell Sci. |
Daxx and histone deacetylase II associate with chromatin through an interaction with core histones and the chromatin-associated protein Dek.
Abstract
- Human Daxx is a protein that functions, in part, as a transcriptional co-repressor through its interaction with a growing number of nuclear, DNA-associated proteins. To determine the mechanism by which hDaxx represses transcription, we used conventional chromatography to isolate endogenous hDaxx. We determined that hDaxx has an apparent molecular weight of 360 kDa, which is consistent with the fact that multiple domains of hDaxx are required for transcriptional repression and suggests that hDaxx associates with multiple proteins. Using co-fractionation and co-immunoprecipitation we demonstrate that hDaxx associates with proteins that are critical for transcriptional repression, such as histone deacetylase II, constituents of chromatin such as core histones H2A, H2B, H3 and H4, and Dek, a chromatin-associated protein reported to change the topology of DNA in chromatin in vitro. We also demonstrate a requirement for the SPT domain and the first paired amphipathic helix of hDaxx for its association with histone deacetylase II and acetylated histone H4, respectively. Finally, we provide evidence suggesting that the association of hDaxx with chromatin-related proteins is dependent on the post-translational phosphorylation status of hDaxx. A working model for the repressive action of hDaxx through its association with chromatin related proteins is presented.
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| 12145313 |
Three-dimensional solution structure of the sodium channel agonist/antagonist delta-conotoxin TxVIA |
10.1074/jbc.M206833200. |
J Biol Chem |
Three-dimensional solution structure of the sodium channel agonist/antagonist delta-conotoxin TxVIA
Abstract
- The three-dimensional solution structure of delta-conotoxin TxVIA, a 27-mer peptide agonist/antagonist of sodium channels, was determined by two-dimensional (1)H NMR spectroscopy with simulated annealing calculations. A total of 20 converged structures of delta-conotoxin TxVIA were obtained on the basis of 360 distance constraints obtained from nuclear Overhauser effect connectivities, 28 torsion angle constraints, and 27 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.35 +/- 0.07 A for the backbone atoms (N, C(alpha), C) and 0.98 +/- 0.14 A for all heavy atoms of the entire peptide. The molecular structure of delta-conotoxin TxVIA is composed of a short triple-stranded antiparallel beta-sheet. The overall beta-sheet topology is +2x, -1, which is the same as those for other conotoxins. However, the three-dimensional structure of delta-conotoxin TxVIA has an unusual hydrophobic patch on one side of the molecule, which may play an important role in the sodium channel binding. These results provide a molecular basis for understanding the mechanism of sodium channel modulation through the toxin-channel interaction and insight into the discrimination of different ion channels.
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