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PubMed1992

Role of Tyr and Trp in membrane responses of Pyrularia thionin determined by optical and NMR spectra following Tyr iodination and Trp modification.

Pyrularia thionin is a strongly basic and bioactive 47 amino acid peptide which contains two Tyr residues at positions 13 and 45 and one Trp at position 8. Limited iodination does not have a significant effect, but prolonged iodination of the peptide leads to progressive inactivation for all known cellular responses (Evans, J. et al. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5849-5853). 1H NMR spectra of the native Pyrularia thionin show four Tyr bands, two arising from each Tyr residue. One resonance band for the epsilon hydrogens of Tyr 45 disappears early during limited iodination and the band from the delta hydrogens shifts to low field. The two bands corresponding to Tyr 13 remain during limited iodination, but both decrease in intensity during prolonged iodination, with the epsilon hydrogen band decreasing somewhat more. The resonance bands arising from Trp disappear during prolonged iodination. This sequence of reactions is verified by the optical absorbance properties of two small peptide fragments obtained by Staphylococcal V8 protease hydrolysis of thionin which had been iodinated to varying degrees. Limited iodination did not significantly inhibit the thionin's biological activity, yet the fragment from the -COOH terminus showed the conversion of Tyr 45 to diiodoTyr. This treatment did not significantly modify the Tyr 13 or Trp 8 located in the -NH2 terminal fragment. More extensive iodination resulted in a disappearance of Trp 8 absorbance with an accompanying conversion of Tyr 13 to the monoiodo form. Extensive iodination yielded two atoms of iodine in the Tyr 45-containing fragment, and only one atom in the Tyr 13 fragment. The data indicate that Tyr 45 of the native thionin is more readily iodinated, proceeding to the diiodo form without significant loss of activity. Prolonged iodination does not lead to the formation of any diiodoTyr 13, but does lead to modification of Trp 8 and probably formation of monoiodoTyr 13. Modification of Trp 8 with N-bromosuccinimide inhibits the hemolytic activity of the thionin, showing that Trp 8 is necessary for Pyrularia thionin activity. It is most likely Trp 8 modification during prolonged iodination which results in the loss of biological activity.

Antimicrobial Cationic Peptides↗

PubMed1988

Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

Azotobacter↗

PubMed1991

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes.

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.

Adult↗

PubMedDate not supplied

The proenkephalin A fragment metorphamide shows supraspinal and spinal opioid activity in vivo.

Metorphamide (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2) a novel amidated octapeptide fragment of proenkephalin A was synthesized, purified and subsequently shown to inhibit the reflex contractions of the rat urinary bladder following intracerebroventricular and spinal intrathecal microinjections. The effects of metorphamide were consistently antagonized by naloxone but not by the delta-opioid receptor antagonist ICI 174,864. Comparison of metorphamide with other proenkephalin A fragments suggested that the activity of this peptide was not due to in vivo processing to other active fragments. These data suggest that metorphamide has potent in vivo mu-opioid activity but little delta-opioid receptor activity.

Amino Acids↗

PubMedDate not supplied

A partial restriction map of the proA-purE region of the Escherichia coli K12 chromosome.

EcoRI restriction mapping data for fragments larger than 0.7 kb and contained in a 350-kb region of the Escherichia coli K-12 chromosome are presented. 75% of these fragments have been located relative to proA, B, argF, lac, proC, purE, and various insertion sequence elements normally present in this region. BglII and BamHI maps for the regions near argF and purE are also provided.

Bacterial Proteins↗

PubMed1995

Cloning of the cDNA encoding human C/EBP gamma, a protein binding to the PRE-I enhancer element of the human interleukin-4 promoter.

The positive regulatory element-I (PRE-I) is a strong enhancer element essential for expression of the human interleukin-4 (IL-4)-encoding gene. In order to identify transcription factors binding to PRE-I, we screened a cDNA expression library from human Jurkat T-cells. A cDNA encoding the human CCAAT/enhancer binding protein-gamma (hC/EBP gamma) was cloned. The deduced amino acid (aa) sequence of HC/EBP gamma contains 150 aa with high homology to mouse Ig/EBP-1 and rat C/EBP gamma. The mRNA of hC/EBP gamma is expressed at a high level in Jurkat T-cells in three forms generated via differential polyadenylation. DNA-binding experiments with recombinant protein produced in bacteria demonstrate that hC/EBP gamma binds to PRE-I, but not to unrelated DNA fragments. Our data also show that hC/EBP gamma may cooperate with Fos to bind PRE-I.

Amino Acid Sequence↗

PubMed1991

High-performance liquid chromatographic-mass spectrometric assay of high-value compounds for pharmaceutical use from plant cell tissue culture: Cinchona alkaloids.

The use of high-performance liquid chromatography (HPLC) interfaced with thermospray (TSP) mass spectrometry is described for the separation and identification of various alkaloids from Cinchona ledgeriana extracts. The use of water-acetonitrile-acetic acid (71:25:4) with 0.01 M ammonium acetate (pH 3.0) as the mobile phase gave good HPLC separation and good TSP sensitivity. The specificity obtained by single-ion monitoring allowed the analysis of commercially important alkaloids such as quinine and quinidine in plant material, transformed roots and in cells from tissue culture, with relatively simple extraction and work-up procedures. TSP gave protonated species with few fragment ions but collision-induced dissociation offers the promise of increased analytically specificity from the fragment ion data. This work has important implications for the biotechnological production of pharmaceuticals normally obtained from plant sources.

Chromatography, High Pressure Liquid↗

PubMed1992

Biostatistical basis of individualization and segregation analysis using the multilocus DNA probe MZ 1.3: results of a collaborative study.

A collaborative study using the multilocus minisatellite DNA probe MZ 1.3 was carried out to investigate segregation information, mutation rate, DNA fragment frequencies as well as band sharing characteristics. The fingerprint patterns of 393 children as well as 694 unrelated individuals were analysed after digestion of DNA with the restriction enzyme HinfI. A mutation rate of 1% per meiosis or 0.04% per band was found with a mean number of 26 bands/individual. It was shown that maternal and paternal fragments are inherited in equal proportions. Population frequencies of restriction fragments demonstrated a distribution with increasing frequencies in the small fragment size range below 10 kb as well as the absence of very common or very rare fragments. Our data can be used to calculate simple exclusion probabilities based on the number of non-maternal bands in the child.

Biometry↗

PubMed2006

Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line.

A proteome map of the most abundant proteins in the murine inner medullary collecting duct (mIMCD3) cell line was generated by 2-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. The 2-D model map identifies 77 distinct constitutive proteins and a total of 86 spots including isoforms. Protein identification was based on both peptide mass fingerprinting (MS) and peptide fragmentation (MS/MS) data. High confidence Mascot scores were obtained in the database search, due to the high quality and the number of MS/MS spectra which provided matching sequence information to the database. A functional classification of the identified proteins showed that a high proportion were stress proteins, such as heat shock proteins and proteins with anti-oxidant activity. Other proteins identified were involved in cytoskeletal maintenance, metabolism and energy generation, as well as in translation, transcription, RNA processing and other cell cycle processes. Exposure of the mIMCD3 cells to hyperosmotic stress using 600 mOsmol/kg NaCl or Urea or 700 mOsmol/kg NaCl-Urea (50:50) resulted in the greatest proteome upregulation in 700 mosM NaCl-Urea and the greatest downregulation in 600 mosM NaCl. Several proteins with molecular chaperone function were induced, such as alpha-B crystallin, two Hsp70 isoforms, the osmotic stress protein (Osp94), as well as aldose reductase. Additional isoforms of the translation elongation factors Eef2 and Eef1a1 were induced. Characterization of the phosphoproteome of mIMCD3 cells with a phosphoprotein-specific stain showed a significant proportion of the proteome was phosphorylated. Additionally, exposure of mIMCD3 cells to 600 mOsmol/kg NaCl hyperosmotic stress resulted in a 1.8-fold higher phosphorylation level of the most acidic isoform of the heat shock protein Hsp27 compared to its phosphorylation level under iso-osmotic conditions.

Aldehyde Reductase↗

PubMed2004

Cytoskeletal actin degradation induced by lovastatin in cardiomyocytes is mediated through caspase-2.

The objective of this study was to test the hypothesis that cytoskeletal actin fragmentation is mediated through caspase-2, specifically examining the ability of a caspase-2 inhibitor to interfere with actin fragmentation, in comparison with a caspase-3 inhibitor. Cardiomyocytes were cultured from embryonic chick heart. The fine structural element of cellular F-actin was visualized by staining cardiomyocytes with NBD-phallacidin. Lovastatin induced a dramatic and concentration-dependent loss of intact F-actin. The selectivity of this effect of lovastatin was demonstrated by the absence of similar changes in F-actin when cardiomyocytes were treated with the apoptotic stimulus palmitate, the metabolism of which produces acetyl CoA, the early substrate of cholesterol synthesis, through the mevalonate pathway. FACS analysis of NBD-phallacidin-stained cells was used to quantify the amount of F-actin loss. Actin fragmentation produced by lovastatin was operative through a caspase-2 pathway, as the caspase-2 inhibitor, z-VDVAD-fmk, significantly blocked lovastatin-induced changes in F-actin, but the caspase-3 inhibitor, Ac-DEVD-CHO, did not. Interruption of the mevalonate pathway was in part responsible for lovastatin's action, as the downstream metabolite mevalonate partially reversed the effect of lovastatin on actin fragmentation. These data indicate a previously unrecognized link between cytoskeletal actin and caspase-2.

Acetyl Coenzyme A↗

PubMed2004

Detection of Helicobacter pylori in bile of cats.

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir.

Animals↗

PubMed2005

Annexin-like alpha giardins: a new cytoskeletal gene family in Giardia lamblia.

Through a genome survey and phylogenetic analysis, we have identified and sequenced 14 new coding regions for alpha-giardins in Giardia lamblia. These proteins are related to annexins and comprise a multi-gene family with 21 members. Many alpha giardins are highly expressed proteins that are very immunogenic during acute giardiasis in humans. However, little is known about the function of these proteins. By using PCR with different combinations of gene-specific primers, we demonstrated that several of the genes localised to the same chromosomal fragment. These data point towards a molecular evolution through gene duplication and subsequent functional divergence. Semi-quantitative reverse transcriptase-PCR analysis of the Giardia life cycle revealed large differences in mRNA expression levels of the alpha giardins. Epitope tagging of the alpha-giardins localised them to different cytoskeletal components, such as the flagella and the adhesive disc, but also to the plasma membrane. These localisation experiments suggest alpha-giardins play a role in cell motility, attachment and membrane stability.

Animals↗

PubMed2004

The characterisation of selected drugs with amine-containing side chains using electrospray ionisation and ion trap mass spectrometry and their determination by HPLC-ESI-MS.

The electrospray ionisation-ion-trap mass spectrometry (ESI-MS(n)) of selected drug compounds with amine-containing side chains has been investigated. Certain characteristic in-source fragmentations have been observed for these molecules. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M + H](+) ions and their predominant fragment ions. These MS(n) experiments also show certain characteristic fragmentations with respect to the amine-containing side chains. QTOF-MS/MS has been used to support the identity of the proposed fragments. The data presented in this paper therefore provides useful information on the structure of these compounds with amine-containing side chains and can be used in the characterisation of such drugs, their structurally related metabolites and unknown molecules of pharmaceutical significance extracted from animal and plant sources, for example. Amphetamine, clenbuterol, flurazepam and methadone can be identified and determined in mixtures at low ng/ml concentrations by the application of HPLC-ESI-MS which can also be used for their analysis in saliva samples.

Amines↗

PubMed2003

Molecular mechanisms of nitric oxide-induced growth arrest and apoptosis in fetal pulmonary arterial smooth muscle cells.

Superoxide plays an important role in pulmonary arterial smooth muscle cell (SMC) proliferation and survival. The rapid reaction between superoxide and nitric oxide (NO) to form peroxynitrite suggests that endothelium-derived NO may influence adjacent SMC growth. To investigate this possibility, we determined the dose-dependent effects of NO on the proliferation and viability of pulmonary arterial SMC isolated from fetal lambs (FPASMC). Using fluorescence microscopy we found a dose-dependent decrease in superoxide levels in FPASMC treated with the NO donor spermine NONOate. This was associated with an increase in peroxynitrite-mediated protein nitration. At doses between 50 and 250 microM, spermine NONOate attenuated serum-induced FPASMC proliferation resulting in a G(0)/G(1) cell cycle arrest. This process involved a decrease in levels of cyclin A and an increase in the nuclear localization of p21 and p27. Furthermore, 500 microM spermine NONOate decreased viable cell number by inducing programmed cell death: FPASMC treated with 500 microM spermine NONOate displayed a loss of mitochondrial membrane potential, followed by caspase activation and DNA fragmentation. These data suggest that NO inhibits superoxide-induced proliferation of FPASMC and at higher doses induces apoptosis. NO donors may therefore prove to be useful therapeutic tools to treat diseases resulting from excessive proliferation of vascular smooth muscle.

Animals↗

PubMed2004

Large-scale genome remodelling by the developmentally programmed elimination of germ line sequences in the ciliate Paramecium.

In Paramecium, during the development of the somatic macronucleus, precise excision of thousands of single-copy non-coding sequences is initiated by specific DNA double-strand breaks, while imprecise elimination of germ-line-limited repeated sequences leads to internal deletions or chromosome fragmentation. Recent data point to a role of non-coding RNAs in the epigenetic programming of these rearrangements.

Animals↗

PubMed2003

Integrin alphavbeta8-mediated activation of transforming growth factor-beta inhibits human airway epithelial proliferation in intact bronchial tissue.

Transforming growth factor (TGF)-beta is a potent multifunctional cytokine that is an essential regulator of epithelial proliferation. Because TGF-beta is expressed almost entirely in a latent state in vivo, a major source of regulation of TGF-beta function is its activation. A subset of integrins, alphavbeta8 and alphavbeta6, which are expressed in the human airway, has recently been shown to activate latent TGF-beta in vitro, suggesting a regulatory role for integrins in TGF-beta function in vivo. Here we have developed a novel, biologically relevant experimental model of human airway epithelium using intact human bronchial tissue. We have used this model to determine the function of integrin-mediated activation of TGF-beta in the airway. In human bronchial fragments cultured in vitro, authentic epithelial-stromal interactions were maintained and integrin and TGF-beta expression profiles correlated with profiles found in normal lung. In addition, in this model, we found that either the integrin alphavbeta8 or TGF-beta could inhibit airway epithelial cell proliferation. Furthermore, we found that one mechanism of integrin-alphavbeta8-dependent inhibition of cell proliferation was through activation of TGF-beta because anti-beta8 antibody blocked the majority (76%) of active TGF-beta released from bronchial fragments. These data provide compelling evidence for a functional role for integrin-mediated activation of TGF-beta in control of human airway epithelial proliferation in vivo.

Aged↗

PubMed1992

Structure of human prostatic acid phosphatase gene.

Two cDNA clones containing the complete protein-coding sequence of 1,188 nucleotides as well as the 5' and 3' non-coding regions of human prostatic acid phosphatase (PAP) were isolated and sequenced. The size of PAP mRNAs from benign prostate hyperplasia and cancerous prostate was estimated to be 3.2Kb, indicating that the 3' downstream polyadenylation signal was used. Several genomic clones containing parts of the human PAP gene were isolated and the nucleotide sequence of ten exons and their flanking regions was determined. The protein-coding sequence of the human PAP gene was interrupted by nine introns. The positions of all nine introns present in the human PAP gene were homologous to those of the first nine introns in the human lysosomal acid phosphatase (LAP) gene. However, the last (11th) exon of the LAP gene encoding the COOH-terminal domain, which includes a transmembrane segment, was found to be absent in human PAP gene. Southern blot analysis of ten mammalian genomic DNAs gave multiple EcoRI fragments. The data of human genomic DNAs were consistent with the total length of the PAP gene of at least 50 kilobases.

Acid Phosphatase↗

PubMed2002

Piceatannol, a Syk-selective tyrosine kinase inhibitor, attenuated antigen challenge of guinea pig airways in vitro.

Activation of nontransmembrane protein tyrosine kinases, such as Lyn and Syk, has been shown to be the earliest detectable signaling response to Fc receptor (Fc epsilon RI) cross-linking on mast cells leading to mast cell degranulation. The present study examined the effects of piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene, 10-100 microM), a Syk-selective tyrosine kinase inhibitor, on ovalbumin-induced anaphylactic contraction of isolated guinea pig bronchi and release of histamine and peptidoleukotrienes from chopped lung preparations. Pretreatment with piceatannol slightly suppressed ovalbumin-induced peak anaphylactic bronchial contraction but markedly (P<0.05) facilitated relaxation of the anaphylactically contracted bronchi. Piceatannol did not inhibit direct histamine-, leukotriene D(4)- or KCl-induced bronchial contraction, nor revert an existing anaphylactic bronchial contraction. Piceatannol, at 30 microM and above, significantly (P<0.05) prevented ovalbumin-induced release of both histamine and peptidoleukotrienes from lung fragments. Piceatannol did not inhibit exogenous arachidonic acid-induced release of peptidoleukotrienes from lung fragments. Our data show for the first time that inhibition of Syk tyrosine kinase can attenuate anaphylactic bronchial contraction in vitro, probably via inhibition of mast cell degranulation.

Airway Obstruction↗