| 12654645 |
In vivo pharmacodynamics of HMR 3270, a glucan synthase inhibitor, in a murine candidiasis model |
10.1128/AAC.47.4.1187-1192.2003. |
Antimicrob Agents Chemother |
In vivo pharmacodynamics of HMR 3270, a glucan synthase inhibitor, in a murine candidiasis model
Abstract
- In vivo pharmacokinetic/pharmacodynamic characterization for numerous antibacterial compounds has had a significant impact upon optimal dosing regimen design and the development of in vivo susceptibility breakpoints. More recently, similar characterization has been undertaken for antifungal drug classes. Very little is known of these pharmacodynamic relationships for the new echinocandin class of compounds. We utilized a neutropenic murine model of disseminated candidiasis to describe the time course antifungal activity of HMR 3270, a new glucan synthase inhibitor. Single-dose in vivo time kill studies with four 16-fold escalating doses demonstrated concentration-dependent killing when drug levels in serum were more than four times the MIC. Postantifungal effects were dose dependent, ranging from 8 to 80 h duration. Multiple dosing regimen studies utilized six total doses, four dosing intervals, and a treatment duration of 6 days. Shortening the dosing interval from every 144 h (q144h) to q36h resulted in a fourfold rise in the dose necessary to achieve a net fungistatic effect. The peak/MIC ratio most strongly correlated with treatment outcomes (peak/MIC ratio, R(2) = 98%; ratio of the area under the concentration-time curve from 0 to 24 h to the MIC, R(2) = 79%, percentage of time above the MIC, R(2) = 61%). Studies were also conducted with five additional Candida albicans isolates to determine if a similar peak/MIC ratio was associated with efficacy. In vivo concentration-dependent killing was similarly observed in studies with each of the additional isolates. The peak/MIC ratio necessary to produce efficacy was relatively similar among the strains studied (P = 0.42). The peak/MIC ratio (mean +/- standard deviation) necessary to achieve a fungistatic effect was 3.72 +/- 1.84, and the ratio necessary to achieve maximal killing was near 10.
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| 12665801 |
Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. |
10.1038/nbt810 |
Nat. Biotechnol. |
Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.
Abstract
- Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step
Results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.
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| 12670868 |
Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell- proliferation factor HCF-1. |
10.1101/gad.252103 |
Genes Dev. |
Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell- proliferation factor HCF-1.
Abstract
- The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription. We show here that separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT). The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated. HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively. Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex. These
Results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure.
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| 12690205 |
Human chromosome 7: DNA sequence and biology. |
10.1126/science.1083423 |
Science |
Human chromosome 7: DNA sequence and biology.
Abstract
- DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.
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| 12691743 |
Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments |
10.1016/s0022-2836(03)00306-1. |
J Mol Biol |
Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments
Abstract
- Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization.
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| 12694387 |
Novel excitatory Conus peptides define a new conotoxin superfamily |
10.1046/j.1471-4159.2003.01685.x. |
J Neurochem |
Novel excitatory Conus peptides define a new conotoxin superfamily
Abstract
- A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.
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| 12695522 |
CCN3 (NOV) is a novel angiogenic regulator of the CCN protein family |
10.1074/jbc.M302028200. |
J Biol Chem |
CCN3 (NOV) is a novel angiogenic regulator of the CCN protein family
Abstract
- CCN3 (NOV) is a matricellular protein of the CCN family, which also includes CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). During development, CCN3 is expressed widely in derivatives of all three germ layers, and high levels of expression are observed in smooth muscle cells of the arterial vessel wall. Altered expression of CCN3 has been observed in a variety of tumors, including hepatocellular carcinomas, Wilm's tumors, Ewing's sarcomas, gliomas, rhabdomyosarcomas, and adrenocortical carcinomas. To understand its biological functions, we have investigated the activities of purified recombinant CCN3. We show that in endothelial cells, CCN3 supports cell adhesion, induces directed cell migration (chemotaxis), and promotes cell survival. Mechanistically, CCN3 supports human umbilical vein endothelial cell adhesion through multiple cell surface receptors, including integrins alphavbeta3, alpha5beta1, alpha6beta1, and heparan sulfate proteoglycans. In contrast, CCN3-induced cell migration is dependent on integrins alphavbeta3 and alpha5beta1, whereas alpha6beta1 does not play a role in this process. Although CCN3 does not contain a RGD sequence, it binds directly to immobilized integrins alphavbeta3 and alpha5beta1, with half-maximal binding occurring at 10 nm and 50 nm CCN3, respectively. Furthermore, CCN3 induces neovascularization when implanted in rat cornea, demonstrating that it is a novel angiogenic inducer. Together, these findings show that CCN3 is a ligand of integrins alphavbeta3 and alpha5beta1, acts directly upon endothelial cells to stimulate pro-angiogenic activities, and induces angiogenesis in vivo.
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| 12704152 |
Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T |
10.1128/IAI.71.5.2775-2786.2003. |
Infect Immun |
Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T
Abstract
- We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.
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| 12707268 |
Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor |
10.1074/jbc.M302425200. |
J Biol Chem |
Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor
Abstract
- Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.
|
| 12724404 |
Identification and characterization of three new components of the mSin3A corepressor complex. |
10.1128/mcb.23.10.3456-3467.2003 |
Mol. Cell. Biol. |
Identification and characterization of three new components of the mSin3A corepressor complex.
Abstract
- The mSin3A corepressor complex contains 7 to 10 tightly associated polypeptides and is utilized by many transcriptional repressors. Much of the corepressor function of mSin3A derives from associations with the histone deacetylases HDAC1 and HDAC2; however, the contributions of the other mSin3A-associated polypeptides remain largely unknown. We have purified an mSin3A complex from K562 erythroleukemia cells and identified three new mSin3A-associated proteins (SAP): SAP180, SAP130, and SAP45. SAP180 is 40% identical to a previously identified mSin3A-associated protein, RBP1. SAP45 is identical to mSDS3, the human ortholog of the SDS3p component of the Saccharomyces cerevisiae Sin3p-Rpd3p corepressor complex. SAP130 does not have detectable homology to other proteins. Coimmunoprecipitation and gel filtration data suggest that the new SAPs are, at the very least, components of the same mSin3A complex. Each new SAP repressed transcription when tethered to DNA. Furthermore, repression correlated with mSin3A binding, suggesting that the new SAPs are components of functional mSin3A corepressor complexes. SAP180 has two repression domains: a C-terminal domain, which interacts with the mSin3A-HDAC complex, and an N-terminal domain, which functions independently of mSin3A-HDAC. SAP130 has a repression domain at its C terminus that interacts with the mSin3A-HDAC complex and an N-terminal domain that probably mediates an interaction with a transcriptional activator. Together, our data suggest that these novel SAPs function in the assembly and/or enzymatic activity of the mSin3A complex or in mediating interactions between the mSin3A complex and other regulatory complexes. Finally, all three SAPs bind to the HDAC-interaction domain (HID) of mSin3A, suggesting that the HID functions as the assembly interface for the mSin3A corepressor complex.
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| 12730668 |
Modulation of p120E4F transcriptional activity by the Gam1 adenoviral early protein. |
10.1038/sj.onc.1206379 |
Oncogene |
Modulation of p120E4F transcriptional activity by the Gam1 adenoviral early protein.
Abstract
- Gam1, an early adenoviral CELO protein, is required for viral replication. Consistent with its ability to inhibit histone deacetylation by HDAC1, Gam1 activates transcription. In this report, we identify the cellular transcription factor p120(E4F) as a Gam1 interaction partner. p120(E4F) is a low-abundance transcription factor that represses the adenovirus E4 promoter. Here we demonstrate that p120(E4F) interacts with HDAC1 in vivo and in vitro, and that E4F-mediated transcriptional repression is alleviated by the HDAC inhibitor trichostatin A or by overexpressing Gam1. A mutant E4 promoter unresponsive to E4F-mediated transcriptional repression is also not stimulated by Gam1. Moreover, our cofractionation experiments demonstrate that p120(E4F), HDAC1 and Gam1 may be concomitantly present in protein complexes. We conclude that Gam1 activates E4-dependent transcription possibly by inactivating HDAC1.
|
| 12731890 |
Characterization of glycosylation sites of the epidermal growth factor receptor |
10.1021/bi027101p. |
Biochemistry |
Characterization of glycosylation sites of the epidermal growth factor receptor
Abstract
- The epidermal growth factor receptor is a transmembrane glycoprotein that mediates the cellular responses to epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). In this study of the human EGF receptor naturally expressed in A431 cells, the glycosylation sites of the full-length, membrane-bound receptor and of a secreted form of the receptor were characterized by mass spectrometry. Our data show that the naturally expressed human EGF receptor is fully glycosylated on eight of the 11 canonical sites; two of the sites are not glycosylated, and one is partially glycosylated, a pattern of site-usage similar but not identical to those reported for the recombinant human EGF receptor heterologously expressed in Chinese hamster ovary cells. We also confirm the partial glycosylation of an atypical NNC site first identified in the receptor expressed in Chinese hamster ovary cells. We show that an additional canonical site in the secreted form of the receptor is fully glycosylated. While the pattern of glycosylation is the same for the sites shared by the full-length and the secreted forms of the receptor, the oligosaccharides of the full-length receptor are more extensively processed. Finally, we provide evidence that in addition to the known secreted form of the receptor, a proteolytic cleavage product of the receptor corresponding to the full extracytoplasmic, ligand-binding domain is present in the conditioned medium.
|
| 12746432 |
Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-L247T nicotinic acetylcholine receptor |
10.1074/jbc.M212628200. |
J Biol Chem |
Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-L247T nicotinic acetylcholine receptor
Abstract
- The effects of the native alpha-conotoxin PnIA, its synthetic derivative [A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L]PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nm, respectively. Rates of onset of inhibition were similar for PnIA and [A10L]PnIA; however, the rate of recovery was slower for [A10L]PnIA, indicating that the increased potency of [A10L]PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nm. In contrast, [A10L]PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [A10L]PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.
|
| 12748394 |
Profiling of cyclic hexadepsipeptides roseotoxins synthesized in vitro and in vivo: a combined tandem mass spectrometry and quantum chemical study |
10.1255/ejms.531. |
Eur J Mass Spectrom (Chichester) |
Profiling of cyclic hexadepsipeptides roseotoxins synthesized in vitro and in vivo: a combined tandem mass spectrometry and quantum chemical study
Abstract
- High-performance liquid chromatography and tandem mass spectrometry (HPLC/MS/MS) was used for the detection of cyclic hexadepsipeptides roseotoxins produced by Trichothecium roseum. Roseotoxins were found in both submerged standard cultivation on CzapekDox medium and in vivo cultivation extract obtained from an apple. Roseotoxin chromatographic profiles from these two experiments were compared. Product-ion collision-induced dissociation (CID) spectra obtained on an ion trap (electrospray ionisation, ESI) were used for the identification of natural roseotoxins A, B, C and of minor destruxins A and B. The dissociation behavior of roseotoxins is discussed in terms of a fragmentation scheme proposed for describing the dissociation pathways of cyclic peptides. This scheme involves opening of the cyclopeptide ring via formation of oxazolone derivatives and fragmentation of the resulting linear species, which have a free N-terminus and an oxazolone ring at the C-terminus. Some aspects of this fragmentation scheme are underlined by modeling the dissociation channels of roseotoxin A using quantum chemical calculations. The structures of roseotoxin A and destruxin B were verified by nuclear magnetic resonance (NMR) spectroscopy. Structures of three new minor natural roseotoxins [Val(4)]RosA, [MeLxx(4)]RosA and [MeLxx(4)]RosB were deduced by ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT-MS) and ion trap tandem mass spectrometry by examining the pre-separated roseotoxin fraction.
|
| 12753377 |
Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach |
10.1034/j.1399-3011.2003.00061.x. |
J Pept Res |
Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach
Abstract
- As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.
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