| 10204851 |
Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis |
None |
J Med Genet |
Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis
Abstract
- Hereditary pancreatitis (HP) is a rare inherited disorder, characterised by recurrent episodes of pancreatitis often beginning in early childhood. The mode of inheritance suggests an autosomal dominant trait with incomplete penetrance. The gene, or at least one of the genes, responsible for hereditary pancreatitis has been mapped to the long arm of chromosome 7 and a missense mutation, an arginine to histidine substitution at residue 117 in the trypsinogen cationic gene (try4) has been shown to segregate with the HP phenotype. The aim of this work was to investigate the molecular basis of hereditary pancreatitis. This study was performed on 14 HP families. The five exons of the trypsinogen cationic gene were studied using a specific gene amplification assay combined with denaturing gradient gel electrophoresis (DGGE). The present paper describes three novel mutations, namely K23R and N29I and a deletion -28delTCC in the promoter region. We also found a polymorphism in exon 4, D162D. In eight of these families we found a mutation which segregates with the disease. A segregation analysis using microsatellite markers carried out on the other families suggests genetic heterogeneity in at least one of them. Our findings confirm the implication of the cationic trypsinogen gene in HP and highlight allelic diversity associated with this phenotype. We also show that the pattern of inheritance of HP is probably complex and that other genes may be involved in this genetic disease.
|
| 10207053 |
Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells |
10.1128/MCB.19.5.3278. |
Mol Cell Biol |
Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells
Abstract
- Insulin-like growth factor II (IGF-II) is a peptide growth factor that is homologous to both insulin-like growth factor I (IGF-I) and insulin and plays an important role in embryonic development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type 1 insulin-like growth factor receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to the alternative splicing of exon 11. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These differences in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal growth and cancer biology.
|
| 10209871 |
Rapid identification of the new anabaenopeptin G from Planktothrix agardhii HUB 011 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry |
10.1002/(SICI)1097-0231(19990315)13:5<337::AID-RCM488>3.0.CO;2-Q. |
Rapid Commun Mass Spectrom |
Rapid identification of the new anabaenopeptin G from Planktothrix agardhii HUB 011 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Abstract
- Toxic water blooms from cyanobacteria in lakes and rivers are a worldwide phenomenon. A new technique is presented for the rapid detection of toxic and nontoxic blooms. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was employed to identify mainly peptide metabolites (microcystins, anabaenopeptins, cyanopeptolins, microviridins, microginins and aeruginosins) from microgram quantities of prepared cells within minutes. The spectra show the presence of peptides in strains or water blooms simultaneously. A new compound has been identified using the post source decay (PSD) and collision induced dissociation (CID) mode. This new compound has been defined as anabaenoeptin G. The potential of the method for screening of various secondary metabolite producers for defined products including antibiotics is discussed.
|
| 10220385 |
Three proteins define a class of human histone deacetylases related to yeast Hda1p. |
10.1073/pnas.96.9.4868 |
Proc. Natl. Acad. Sci. U.S.A. |
Three proteins define a class of human histone deacetylases related to yeast Hda1p.
Abstract
- Gene expression is in part controlled by chromatin remodeling factors and the acetylation state of nucleosomal histones. The latter process is regulated by histone acetyltransferases and histone deacetylases (HDACs). Previously, three human and five yeast HDAC enzymes had been identified. These can be categorized into two classes: the first class represented by yeast Rpd3-like proteins and the second by yeast Hda1-like proteins. Human HDAC1, HDAC2, and HDAC3 proteins are members of the first class, whereas no class II human HDAC proteins had been identified. The amino acid sequence of Hda1p was used to search the GenBank/expressed sequence tag databases to identify partial sequences from three putative class II human HDAC proteins. The corresponding full-length cDNAs were cloned and defined as HDAC4, HDAC5, and HDAC6. These proteins possess certain features present in the conserved catalytic domains of class I human HDACs, but also contain additional sequence domains. Interestingly, HDAC6 contains an internal duplication of two catalytic domains, which appear to function independently of each other. These class II HDAC proteins have differential mRNA expression in human tissues and possess in vitro HDAC activity that is inhibited by trichostatin A. Coimmunoprecipitation experiments indicate that these HDAC proteins are not components of the previously identified HDAC1 and HDAC2 NRD and mSin3A complexes. However, HDAC4 and HDAC5 associate with HDAC3 in vivo. This finding suggests that the human class II HDAC enzymes may function in cellular processes distinct from those of HDAC1 and HDAC2.
|
| 10223951 |
Improved derivatives of bactenecin, a cyclic dodecameric antimicrobial cationic peptide |
10.1128/AAC.43.5.1274. |
Antimicrob Agents Chemother |
Improved derivatives of bactenecin, a cyclic dodecameric antimicrobial cationic peptide
Abstract
- Both linear and cyclic derivatives of the cyclic 12-amino-acid antimicrobial peptide bactenecin were designed based on optimization of amphipathicity and charge location. In general, increasing the number of positive charges at the N and C termini and adding an extra tryptophan residue in the loop not only increased the activities against both gram-positive and gram-negative bacteria but also broadened the antimicrobial spectrum.
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| 10224016 |
Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900 |
10.1128/AEM.65.5.2170-2178.1999. |
Appl Environ Microbiol |
Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900
Abstract
- A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287-2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.
|
| 10228163 |
Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis |
10.1093/emboj/18.9.2489. |
EMBO J |
Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis
Abstract
- Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.
|
| 10233432 |
Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia |
None |
Br J Haematol |
Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia
Abstract
- Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.
|
| 10318918 |
NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling |
10.1073/pnas.96.10.5533. |
Proc Natl Acad Sci U S A |
NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling
Abstract
- Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1/2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.
|
| 10319747 |
Comparison of 111In-DOTA-Tyr3-octreotide and 111In-DTPA-octreotide in the same patients: biodistribution, kinetics, organ and tumor uptake |
None |
J Nucl Med |
Comparison of 111In-DOTA-Tyr3-octreotide and 111In-DTPA-octreotide in the same patients: biodistribution, kinetics, organ and tumor uptake
Abstract
- Scintigraphy with 111In-diethylenetriamine pentaacetic acid0-D-Phe1-octreotide (DTPAOC) is used to demonstrate neuroendocrine and other somatostatin-receptor-positive tumors. Despite encouraging results, this 111In-labeled compound is not well suited for peptide-receptor-mediated radiotherapy of somatostatin-receptor-positive tumors. Another somatostatin analog, 1,4,7,10-tetraazacyclododecane-N,N\,N",N\\\-tetraacetic acid0, D-Phe1, Tyr3-octreotide (DOTATOC), can be labeled with the beta-emitter 90Y in a stable manner.\n \n \n \n \n We compared the distribution, kinetics and dosimetry of 111In-DTPAOC and 111In-DOTATOC in eight patients to predict the outcomes of these parameters in patients who will be treated with 90Y-DOTATOC.\n \n \n \n \n Serum radioactivity levels for the radiopharmaceuticals did not differ significantly 2-24 h after injection (P>0.05). Up to 2 h postinjection they were slightly, but significantly, lower after administration of 111In-DOTATOC (P < 0.01 at most time points). The percentage of peptide-bound radioactivity in serum did not differ after administration of either compound. Urinary excretion was significantly lower after administration of 111In-DOTATOC (P < 0.01). The visualization of known somatostatin-receptor-positive organs and tumors was clearer after administration of 111In-DOTATOC than after administration of 111In-DTPAOC. This was confirmed by significantly higher calculated uptakes in the pituitary gland and spleen. The uptake in the tumor sites did not differ significantly (P > 0.05), although in three of the four patients in whom tumor uptake could be calculated, it was higher after administration of 111In-DOTATOC.\n \n \n \n \n The distribution and excretion pattern of 111In-DOTATOC resembles that of 111In-DTPAOC, and the uptake in somatostatin-receptor-positive organs and most tumors is higher for 111In-DOTATOC. If 90Y-DOTATOC shows an uptake pattern similar to 111In-DOTATOC, it is a promising radiopharmaceutical for peptide-receptor-mediated radiotherapy in patients with somatostatin-receptor-positive tumors.
|
| 10320362 |
Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors |
10.1021/bi982817z. |
Biochemistry |
Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors
Abstract
- Two novel alpha-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main alpha-conotoxin contained in the venom, whereas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 microM which can be compared to the IC50 of 0.31 microM found for the previously characterized alpha-MI isolated from the piscivorous Conus magus. The synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for alpha-GI and alpha-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 degrees C.
|
| 10329447 |
Structural basis for the binding specificity of a SSTR1-selective analog of somatostatin |
10.1006/bbrc.1999.0699. |
Biochem Biophys Res Commun |
Structural basis for the binding specificity of a SSTR1-selective analog of somatostatin
Abstract
- The availability of subtype-specific agonists and antagonists for somatostatin (SS) receptors (SSTRs) will be important for elucidation of the function of each receptor isoform in vivo. A SS analog, des-AA1,2,5-D-Trp8, IAmp9SS (CH275), has been shown previously to bind preferentially to SSTR1. In this report, we identify structural determinants in the ligand and receptor responsible for the selective binding of CH275 to SSTR1 by modifying both the ligand and the receptor. We propose that IAmp9 in CH275, like Lys9 in SS, interacts with Asp137 in the middle of the third transmembrane domain of SSTR1 to form an ion pair, while other residues unique to SSTR1 conbribute to binding selectivity of CH275 for SSTR1. Replacement of Asp137 with Asn resulted in loss of binding of radiolabeled SS and decreased potencies of both SS and CH275 to induce a change in the extracellular acidification rate measured by microphysiometry. The structural determinants for specific binding to SSTR1 were mapped in chimeric SSTR1/SSTR2 receptors. One chimera, 2beta, with the N-terminus to second transmembrane domain (TM2) from SSTR2 and the remainder of the receptor from SSTR1, had low affinity for CH275. Furthermore, when a single residue, Leu107, in TM2 of SSTR1 was replaced with Phe, the corresponding residue in SSTR2, a 20-fold decrease in affinity for CH275 with no significant change in affinity for SS was observed. A reciprocal change from Phe to Leu in the chimeric receptor 2beta resulted in a 10-fold increase in affinity for CH275. Thus, Leu107 is an important determinant for CH275 binding to SSTR1. To identify the moiety in CH275 which could interact with Leu107, a new analog des-AA1,2,5-D-Trp8, Amp9SS was prepared. This analog bound to both SSTR1 and SSTR2 with similar affinities; thus, subtype selectivity was lost. Collectively, these data support a binding model for CH275 in which the positively charged IAmp interacts with the negatively charged Asp137 in TM3 of SSTR1 and the isopropyl group of IAmp forms a hydrophobic interaction with Leu107 in TM2.
|
| 10340607 |
Anabaenopeptins G and H, potent carboxypeptidase A inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-595) |
10.1016/s0960-894x(99)00191-2. |
Bioorg Med Chem Lett |
Anabaenopeptins G and H, potent carboxypeptidase A inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-595)
Abstract
- Anahaenopeptins G (1) and H (2) were isolated from the cultured cyanobacterium Oscillatoria agardhii (NIES-595) as potent carboxypeptidase A (CPA) inhibitors. The gross structure of 1 and 2 were established by spectroscopic analysis including the 2D NMR technique and the absolute configurations of 1 and 2 were determined by the spectral and chemical methods. 1 and 2 inhibited CPA with IC50's of 0.0018 and 3.4 microg/mL, respectively."
|
| 10346923 |
Solution conformation of a potent cyclic analogue of tuftsin: low- temperature nuclear magnetic resonance study in a cryoprotective mixture |
10.1021/jm980442+. |
J Med Chem |
Solution conformation of a potent cyclic analogue of tuftsin: low- temperature nuclear magnetic resonance study in a cryoprotective mixture
Abstract
- Tuftsin, a linear tetrapeptide (Thr-Lys-Pro-Arg), corresponding to the sequence 289-292 of the heavy chain of leukokinin, has been the object of intensive SAR studies during the past 30 years, owing to its numerous biological activities and to the possibility of generating a novel anticancer drug. A cyclic tuftsin analogue, c-T-K-P-R-G, has biological activity 50 times higher than that of the parent linear peptide. Here we present a conformational study of c-T-K-P-R-G based on NMR data in a cryoprotective DMSO/water mixture. The preferred conformation is a type VIa turn centered on the K-P residues. The orientation of the side chains of the two basic residues (K and R) may represent the essential feature of the bioactive conformation of tuftsin. A possible role of tuftsin as a DNA binding motif is suggested by the similarity of the bioactive conformation of c-T-K-P-R-G and of the beta-turn conformation proposed by Suzuki for the T,S-P-K-R motif.
|
| 10347882 |
Comparative toxicity of four microcystins of different hydrophobicities to the protozoan, Tetrahymena pyriformis |
10.1046/j.1365-2672.1999.00771.x. |
J Appl Microbiol |
Comparative toxicity of four microcystins of different hydrophobicities to the protozoan, Tetrahymena pyriformis
Abstract
- Microcystins (MC) are a group of over 60 cyclic heptapeptide hepatotoxins produced by cyanobacteria. The 1-octanol/water partition coefficients (log P) of MC-LR, -LY, -LW and -LF have been estimated by HPLC to be 2.16, 2.92, 3.46 and 3.56, respectively. Their in vivo toxicities to Tetrahymena pyriformis was also investigated. Twenty-four hour LC50 values followed the order MC-LR > -LY > -LW approximately -LF. The LC50 values of MC-LR and -LY were significantly reduced in the presence of 1% (v/v) dimethylsulphoxide, although no significant effect occurred with MC-LW or -LF. Tetrahymena pyriformis respiration rates were inhibited by MC-LR in both a time- and dose-dependent manner. Increasing log P of the MC used caused a significantly greater inhibition of respiration. Population growth rate and maximum culture density were inhibited by all MC variants in proportion to log P. Positive correlations between all toxicological endpoints and log P occurred, with the most hydrophobic toxin, MC-LF, being 1.4 to 3.5 times more toxic than MC-LR. MC-LW had a similar toxicity to MC-LF, while MC-LY toxicity was intermediate between that of MC-LR and -LF. Implications of this positive relationship between in vivo toxicity and hydrophobicity for the toxicity of MC to aquatic organisms, and the potential for using log P as a descriptor in a quantitative structure-activity relationship for MC, are discussed.
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