| 10521339 |
A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins |
10.1126/science.286.5439.498. |
Science |
A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins
Abstract
- Analysis of rhesus macaque leukocytes disclosed the presence of an 18-residue macrocyclic, tridisulfide antibiotic peptide in granules of neutrophils and monocytes. The peptide, termed rhesus theta defensin-1 (RTD-1), is microbicidal for bacteria and fungi at low micromolar concentrations. Antibacterial activity of the cyclic peptide was threefold greater than that of an open-chain analog, and the cyclic conformation was required for antimicrobial activity in the presence of 150 millimolar sodium chloride. Biosynthesis of RTD-1 involves the head-to-tail ligation of two alpha-defensin-related nonapeptides, requiring the formation of two new peptide bonds. Thus, host defense cells possess mechanisms for synthesis and granular packaging of macrocyclic antibiotic peptides that are components of the phagocyte antimicrobial armamentarium.
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| 10521717 |
Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly] |
None |
Biochemistry (Mosc) |
Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly]
Abstract
- A biologically active analog of beta-casomorphin, H-Tyr-cyclo[D-OrnPheProGly], was studied by theoretical conformational analysis. Random sampling was used to search the conformational space of allowed dihedral angles. Among 53 low-energy conformers with different folding of the peptide cyclic moiety, only those were selected which correspond to the low-energy area of the model linear tripeptide Ac-D-AlaAlaPro-NHMe. This peptide was used as the main chain "template" for the D-OrnPheProGly fragment of the studied cyclopeptide molecule. Only 15 conformers were chosen as potentially biologically active structures. The conformational possibilities of the Phe residue were constrained to the combined (A + G) region of the Ala residue phi,psi-map for linear peptides.
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| 10523301 |
Cell-type specific phosphorylation of threonines T654 and T669 by PKD defines the signal capacity of the EGF receptor |
10.1093/emboj/18.20.5567. |
EMBO J |
Cell-type specific phosphorylation of threonines T654 and T669 by PKD defines the signal capacity of the EGF receptor
Abstract
- In Rat-1 fibroblasts epidermal growth factor (EGF), but not platelet-derived growth factor (PDGF) stimulates the activity of the c-Jun N-terminal kinase (JNK). Moreover, PDGF induced suppression of EGF-mediated JNK activation, apparently through protein kinase C (PKC) activation. Further analysis revealed that PKD was specifically activated by PDGF but not EGF in Rat-1 cells. In SF126 glioblastoma cells, however, EGF and PDGF synergistically activated JNK, while neither PDGF nor EGF stimulated PKD activity. In this cell line, overexpression of PKD blocked EGF- and PDGF-induced JNK activation. Mutational analysis further revealed that the EGFR mutant (T654/669E) was incapable of activating JNK and provided evidence that PKD-mediated dual phosphorylation of these critical threonine residues leads to suppression of EGF-induced JNK activation. Our results establish a novel crosstalk mechanism which allows signal integration and definition in cells with many different RTKs.
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| 10523670 |
HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor. |
10.1128/mcb.19.11.7816 |
Mol. Cell. Biol. |
HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor.
Abstract
- Histone acetylation plays an important role in regulating chromatin structure and thus gene expression. Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37. HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain. When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain. Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These
Results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.
|
| 10543955 |
MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins |
10.1006/jmbi.1999.3163. |
J Mol Biol |
MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins
Abstract
- MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.
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| 10545176 |
Single-residue alteration in alpha-conotoxin PnIA switches its nAChR subtype selectivity |
10.1021/bi991252j. |
Biochemistry |
Single-residue alteration in alpha-conotoxin PnIA switches its nAChR subtype selectivity
Abstract
- alpha-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different alpha-conotoxins are able to discriminate among different subtypes of mammalian nAChRs. In this report, the activity of two peptides from the venom of Conus pennaceus, alpha-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks alpha3beta2 nAChRs, whereas PnIB prefers the alpha7 subtype. Point mutation chimeras of these alpha-conotoxins were synthesized and their activities assessed on Xenopus oocytes expressing specific nAChRs. Change of a single residue, Ala10 to Leu, in PnIA (to form PnIA [A10L]) converts the parent peptide from alpha3beta2-preferring to alpha7-preferring; furthermore, PnIA [A10L] blocks the alpha7 receptor with an IC(50) (12.6 nM) that is lower than that of either parent peptide. Kinetic analysis indicates that differences in affinity among the analogues are primarily due to differences in off-rate, with PnIA [A10L]'s interaction with alpha7 having the smallest off-rate (k(off) = 0.17 min(-)(1)). Thermodynamic analysis indicates that Leu10 enhances the peptide's interaction with alpha7, but not alpha3beta2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both alpha7 and alpha3beta2 nAChRs.
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| 10545197 |
Molecular association between ATR and two components of the nucleosome remodeling and deacetylating complex, HDAC2 and CHD4. |
10.1021/bi991614n |
Biochemistry |
Molecular association between ATR and two components of the nucleosome remodeling and deacetylating complex, HDAC2 and CHD4.
Abstract
- Ataxia telangiectasia mutated (ATM)- and Rad3-related protein (ATR) is a phosphatidylinositol-kinase (PIK)-related kinase that has been implicated in the response of human cells to multiple forms of DNA damage and may play a role in the DNA replication checkpoint. The purification of an ATR complex allowed identification of chromodomain-helicase-DNA-binding protein 4 (CHD4) as an ATR-associated protein by tandem mass spectrometric sequencing. CHD4 (also called Mi-2beta) is a component of a histone-deacetylase-2 (HDAC2)-containing complex, the nucleosome remodeling and deacetylating (NRD) complex. Endogenous ATR, CHD4, and HDAC2 are shown to coimmunoprecipitate, and ATR and HDAC2 coelute through two biochemical purification steps. Other members of the NRD complex, HDAC1, MTA1, and MTA2, are also detectable in ATR immunoprecipitates. ATR's association with CHD4 and HDAC2 suggests that there may be a linkage between ATR's role in mediating checkpoints induced by DNA damage and chromatin modulation via remodeling and deacetylation.
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| 10550770 |
Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity |
10.1002/(sici)1097-0290(19991220)65:6<638::aid-bit4>3.0.co;2-k. |
Biotechnol Bioeng |
Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity
Abstract
- Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).
|
| 10551778 |
New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype. |
10.1210/mend.13.11.0370 |
Mol. Endocrinol. |
New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype.
Abstract
- Premature ovarian failure occurs in almost 1% of women under age 40. Molecular alterations of the FSH receptor (FSHR) have recently been described. A first homozygous mutation of the FSHR was identified in Finland. More recently, we described two new mutations of the FSHR in a woman presenting a partial FSH-resistance syndrome (patient 1). We now report new molecular alterations of the FSHR in another woman (patient 2) who presented at the age of 19 with primary amenorrhea contrasting with normal pubertal development. She had high plasma FSH, and numerous ovarian follicles up to 3 mm in size were evidenced by ultrasonography. Histological and immunohistochemical examination of ovarian biopsies revealed the presence of a normal follicular development up to the antral stage and disruption at further stages. DNA sequencing showed two heterozygous mutations: Asp224Val in the extracellular domain and Leu601Val in the third extracellular loop of FSHR. Cells transfected with expression vectors encoding the wild type or the mutated Leu601Val receptors bound hormone with similar affinity, whereas binding was barely detectable with the Asp224Val mutant. Confocal microscopy showed the latter to have an impaired targeting to the cell membrane. This was confirmed by its accumulation as a mannose-rich precursor. Adenylate cyclase stimulation by FSH of the Leu601Val mutant receptor showed a 12+/-3% residual activity, whereas in patient 1 a 24+/-4% residual activity was detected for the Arg573Cys mutant receptor. These
Results are in keeping with the fact that estradiol and inhibin B levels were higher in patient 1 and that stimulation with recombinant FSH did not increase follicular size, estradiol, or inhibin B levels in patient 2 in contrast to what was observed for patient 1. Thus, differences in the residual activity of mutated FSHR led to differences in the clinical, biological, and histological phenotypes of the patient.
|
| 10551867 |
Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain. |
10.1074/jbc.274.46.32997 |
J. Biol. Chem. |
Somatostatin receptor interacting protein defines a novel family of multidomain proteins present in human and rodent brain.
Abstract
- By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile alpha motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.
|
| 10554184 |
Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol |
10.1002/(sici)1097-0320(19990101)35:1<80::aid-cyto11>3.3.co;2-#. |
Cytometry |
Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol
Abstract
- Techniques to measure apoptosis are used to study a wide spectrum of conditions, from acquired immune deficiency syndrome (AIDS) to cancer to autoimmune diseases. Therefore, a critical comparison of common assays for apoptosis is warranted.
The kinetics of apoptosis induction in dexamethasone (DEX)-exposed thymocytes was examined after 2, 4, 8, 12, 26-28, and 48-50 h of culture. An additional aim was to ascertain whether a similar thymic atrophy-inducing hormone, diethylstilbestrol (DES), also directly induces thymocyte apoptosis. Apoptosis was evaluated by flow cytometric examination of cells stained with propidium iodide (PI), 7-aminoactinomycin D (7-AAD), or fluorescein isothiocyante (FITC)-annexin; by forward-and side-scatter (FS, SS) analysis, cell-size analyzer; and through cytopathologic examination.
After 4 h of DEX exposure, apoptosis was evident by 7-AAD, annexin, and cytopathological assays, but no cells with sub-diploid DNA content were evident by PI analysis. Maximal apoptosis was evident by all the above flow cytometric techniques at 12 h after DEX exposure. The 7-AAD and annexin assays, which allow discrimination between early apoptosis and late apoptosis/necrosis, were comparable and identified similar apoptotic populations. Appearance of a FSlow/SSincreased population was evident only after 12 h of DEX exposure. Apoptosis could not be detected by any of the above assays in thymocytes exposed to various doses of DES.
Two of the six assays, 7-AAD and annexin, were similar in detecting apoptosis at an early kinetic time point. Results of both assays were comparable at all time points studied. Our studies imply that DEX and DES induce thymic atrophy, in vivo, by different mechanisms.
|
| 10556215 |
The t(11;20)(p15;q11) chromosomal translocation associated with therapy- related myelodysplastic syndrome results in an NUP98-TOP1 fusion. |
10.1073/pnas.86.21.8492 |
Blood |
The t(11;20)(p15;q11) chromosomal translocation associated with therapy- related myelodysplastic syndrome results in an NUP98-TOP1 fusion.
Abstract
- The NUP98 gene is involved in 3 distinct chromosomal rearrangements, t(7;11)(p15;p15), t(2;11)(q31;p15), and inv(11)(p15q22); all of these NUP98 rearrangements have been identified in the malignant cells of patients with therapy-related acute myelogenous leukemia or myelodysplastic syndrome (t-AML/MDS). Here we report the cloning and characterization of a t(11;20)(p15;q11) translocation from patients with t-MDS. The breakpoint on chromosome 11p15 targets the NUP98 gene and
Results in the separation of the N-terminal FXFG repeats from the RNA-binding domain located in the C-terminus. The breakpoint on chromosome 20q11 occurs within the gene encoding human DNA topoisomerase I (TOP1). As a result, a chimeric mRNA encoding the NUP98 FXFG repeats fused to the body of DNA topoisomerase I is produced. These
Results indicate that NUP98 is a recurrent target in therapy-related malignancies, and that TOP1 is a previously unrecognized target for chromosomal translocations.
|
| 10557314 |
The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase |
10.1073/pnas.96.23.13294. |
Proc Natl Acad Sci U S A |
The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase
Abstract
- Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a beta-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.
|
| 10561573 |
Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp |
10.1046/j.1432-1327.1999.00855.x. |
Eur J Biochem |
Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp
Abstract
- Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.
|
| 10561589 |
Conformations in solution of the fuscopeptins. Phytotoxic metabolites of Pseudomonas fuscovaginae |
10.1046/j.1432-1327.1999.00883.x. |
Eur J Biochem |
Conformations in solution of the fuscopeptins. Phytotoxic metabolites of Pseudomonas fuscovaginae
Abstract
- Fuscopeptins are phytotoxic amphiphilic lipodepsipeptides containing 19 amino acid residues. They are produced by the plant pathogenic bacterium Pseudomonas fuscovaginae in two forms, A and B, which differ only in the number of methylene groups in the fatty acid chain. Their covalent structure and biological properties have been reported previously. CD and NMR spectroscopy investigations in solution revealed the absence of identifiable elements of secondary and tertiary structure for these molecules. Fuscopeptin B appears to be completely unstructured in aqueous solution, and has a large molecular flexibility. A dramatic conformational change was observed upon addition of trifluoroethanol. This study reports the complete interpretation of the two-dimensional NMR spectra and the NOE results obtained for fuscopeptin B in water/trifluoroethanol solutions; the signals relative to the peptidic moiety are identical to those observed for fuscopeptin A. The results of this investigation were used to determine the solution structure of fuscopeptin B by computer simulations applying distance geometry and simulated annealing procedures. In water/trifluoroethanol solutions the peptidic region appears to have a partly helical structure. The lactonic ring assumes defined conformations very similar to those already reported for other lipodepsipeptides. The structure for fuscopeptin B in solution is also valid for fuscopeptin A because of the negligible structural difference between the two metabolites.
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