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U Thomas

Publications and source records attributed to U Thomas.

At least 19 recordsLinked to original sources
PubMed2001

A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action.

Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98-112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loop-helix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.

Amino Acid Sequence↗
PubMed2001

Isolation and characterization of four bactericidal domains in the bovine beta-lactoglobulin.

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.

Amino Acid Sequence↗
PubMed2000

Synaptic targeting and localization of discs-large is a stepwise process controlled by different domains of the protein.

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs) assemble ion channels, cell-adhesion molecules and components of second messenger cascades into synapses, and are therefore potentially important for co-ordinating synaptic strength and structure. Here, we have examined the targeting of the Drosophila MAGUK Discs-large (DLG) to larval neuromuscular junctions. RESULTS: During development, DLG was first found associated with the muscle subcortical compartment and plasma membrane, and later was recruited to the postsynaptic membrane. Using a transgenic approach, we studied how mutations in various domains of the DLGprotein affect DLG targeting. Deletion of the HOOK region-the region between the Src homology 3 (SH3) domain and the guanylate-kinase-like (GUK) domain-prevented association of DLG with the subcortical network and rendered the protein largely diffuse. Loss of the first two PDZ domains led to the formation of large clusters throughout the plasma membrane, with scant targeting to the neuromuscular junction. Proper trafficking of DLG missing the GUK domain depended on the presence of endogenous DLG. CONCLUSIONS: Postsynaptic targeting of DLG requires a HOOK-dependent association with extrasynaptic compartments, and interactions mediated by the first two PDZ domains. The GUK domain routes DLG between compartments, possibly by interacting with recently identified cytoskeletal-binding partners.

Animals↗
PubMed2000

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli.

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.

Anti-Bacterial Agents↗
PubMed1999

Regulation of DLG localization at synapses by CaMKII-dependent phosphorylation.

Discs large (DLG) mediates the clustering of synaptic molecules. Here we demonstrate that synaptic localization of DLG itself is regulated by CaMKII. We show that DLG and CaMKII colocalize at synapses and exist in the same protein complex. Constitutively activated CaMKII phenocopied structural abnormalities of dlg mutant synapses and dramatically increased extrajunctional DLG. Decreased CaMKII activity caused opposite alterations. In vitro, CaMKII phosphorylated a DLG fragment with a stoichiometry close to one. Moreover, expression of site-directed dlg mutants that blocked or mimicked phosphorylation had effects similar to those observed upon inhibiting or constitutively activating CaMKII. We propose that CaMKII-dependent DLG phosphorylation regulates the association of DLG with the synaptic complex during development and plasticity, thus providing a link between synaptic activity and structure.

Animals↗
PubMed1999

Isolation and identification of three bactericidal domains in the bovine alpha-lactalbumin molecule.

Proteolytic digestion of alpha-lactalbumin by pepsin, trypsin and chymotrypsin yielded three polypeptide fragments with bactericidal properties. Two fragments were obtained from the tryptic digestion. One was a pentapeptide with the sequence EQLTK (residues 1-5) and the other, GYGGVSLPEWVCTTF ALCSEK (residues (17-31)S-S(109-114)), was composed of two polypeptide chains held together by a disulfide bridge. Fragmentation of alpha-lactalbumin by chymotrypsin yielded CKDDQNPH ISCDKF (residues (61-68)S-S(75-80)), also a polypeptide composed of two polypeptide chains held together by a disulfide bridge. The three polypeptides were synthesized and found to exert antimicrobial activities. The polypeptides were mostly active against Gram-positive bacteria. Gram-negative bacteria were only poorly susceptible to the bactericidal action of the polypeptides. GYGGVSLPEWVCTTF ALCSEK was most, EQLTK least bactericidal. Replacement of leucine (23) with isoleucine, having a similar chemical structure but higher hydrophobicity, in the sequence GYGGVSLPEWVCTTF ALCSEK significantly reduced the bactericidal capacity of the polypeptide. Digestion of alpha-lactalbumin by pepsin yielded several polypeptide fragments without antibacterial activity. alpha-Lactalbumin in contrast to its polypeptide fragments was not bactericidal against all the bacterial strains tested. Our results suggest a possible antimicrobial function of alpha-lactalbumin after its partial digestion by endopeptidases.

Amino Acid Sequence↗
PubMed1997

Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution.

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Escherichia coli↗
PubMed1997

Synaptic clustering of the cell adhesion molecule fasciclin II by discs-large and its role in the regulation of presynaptic structure.

The cell adhesion molecule Fasciclin II (FASII) is involved in synapse development and plasticity. Here we provide genetic and biochemical evidence that proper localization of FASII at type I glutamatergic synapses of the Drosophila neuromuscular junction is mediated by binding between the intracellular tSXV bearing C-terminal tail of FASII and the PDZ1-2 domains of Discs-Large (DLG). Moreover, mutations in fasII and/or dlg have similar effects on presynaptic ultrastructure, suggesting their functional involvement in a common developmental pathway. DLG can directly mediate a biochemical complex and a macroscopic cluster of FASII and Shaker K+ channels in heterologous cells. These results indicate a central role for DLG in the structural organization and downstream signaling mechanisms of cell adhesion molecules and ion channels at synapses.

Animals↗
PubMed1997

Functional expression of rat synapse-associated proteins SAP97 and SAP102 in Drosophila dlg-1 mutants: effects on tumor suppression and synaptic bouton structure.

The synapse-associated proteins SAP97 and SAP102 are mammalian proteins that are structurally related to the Drosophila tumor suppressor protein DlgA. Previous analyses revealed that DlgA is essential for the integrity of epithelia and neuromuscular synapses. Here we show that synaptic bouton structure is severely affected in mutant larvae carrying the dlg-1(XI-2) allele. We have tested SAP97 and SAP102 for functional homology to DlgA by heterologous expression in Drosophila. Both SAP97 and SAP102 can suppress tumor formation in dlg-1 mutant flies and mimic DlgA at larval neuromuscular junctions. Neuronal expression of SAP97 or SAP102 is required for morphological restoration of synaptic boutons, indicating that presynaptic DlgA function is essential for establishing structurally intact motor nerve terminals at larval neuromuscular junctions.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Identification and isolation of a bactericidal domain in chicken egg white lysozyme.

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria. Fractionation of clostripain-digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I-V-S-D-G-N-G-M-N-A-W-V-A-W-R (amino acids 98-112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I-V-S-D-G-N-G-M-N-A-W-V-A-W-R. The peptide I-V-S-D-G-N-G-M consisting of the eight amino acids of the N-terminal side had no bactericidal properties, whereas the peptide N-A-W-V-A-W-R of the C-terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R-A-W-V-A-W-R) increased the bactericidal activity considerably. The D enantiomer of R-A-W-V-A-W-R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.

Animals↗
PubMed1996

Identification and isolation of the bactericidal domains in the proteinase inhibitor aprotinin.

Aprotinin is a protein proteinase inhibitor of 58 amino acids, located in bovine mast cells which also possesses antibacterial activity. Digestion of aprotinin by clostripain yielded three antimicrobial oligopeptide fragments with the sequences RPDFCLEPPYTGPCK (residues 1-15), IIRYFYNAKAGLCQTFVYGGCR (residues 18-39) and AKRNNFKSAEDCMRTCGGA (residues 40-58). With the exception of residues 16 (alanyl) and 17 (arginyl) they cover the whole aprotinin structure. The three oligopeptides were synthesized and found to exert a broad spectrum of antimicrobial activities. Most potent was oligopeptide IIRYFYNAKAGLCQTFVYGGCR which at a concentration of 7 x 10(-9) M exhibited a high bactericidal activity against the eleven gram-positive and gram-negative bacterial strains tested. None of the purified oligopeptides showed any antiproteolytic activity. Our results suggest that aprotinin has multiple antimicrobial domains which are independent of the antiproteolytic function of the original molecule.

Amino Acid Sequence↗
PubMed1996

Effect of ischemic preconditioning of the myocardium on cAMP.

Reduction of cAMP has been implicated in the protection of ischemic preconditioning (IP), but until now, this possibility has not been directly addressed. In this study, we found that in the in vivo rabbit heart 10 to 30 minutes of sustained regional ischemia was accompanied by a nearly twofold rise in cAMP levels. This increase in cAMP was attenuated when sustained ischemia was preceded by IP induced with a single cycle of transient ischemia and reperfusion (TI/R) and prevented when ischemia was preceded by three cycles of TI/R. The mechanism of cAMP reduction by IP does not involve activation of protein kinase C (PKC), since the PKC inhibitor polymyxin B (24 mg/kg) did not raise cAMP levels during sustained ischemia in IP hearts. Furthermore, this effect is also not mediated by reduced responsiveness of the beta-adrenergic effector pathway, since both nonischemic hearts and hearts subjected to three cycles of TI/R exhibited similar increases in cAMP in response to 5 micrograms/kg isoproterenol. However, propranolol (0.75 mg/kg) abolished the rise in cAMP levels observed during sustained ischemia in control hearts but did not reduce cAMP levels further in IP hearts. These data indicate that the ischemia-induced rise in cAMP levels in control hearts was mediated by activation of the beta-adrenergic receptor. Taken together with data demonstrating that beta-adrenergic responsiveness was not affected by IP, these data support the conclusion that the lack of elevation in cAMP levels observed during sustained ischemia in IP hearts is mediated by an attenuation of norepinephrine release. To examine whether the protection of IP against necrosis was mediated by the lack of elevation in cAmp levels, we determined whether the infarct size-limiting effect of IP could be blocked by NKH477, an activator of adenylyl cyclase. Four groups or rabbits were subjected to 30 minutes of in vivo regional ischemia and 90 minutes of reperfusion. Control hearts (n = 10) had 53.6 +/- 5.5% infarction of the area at risk. IP with three cycles of transient ischemia limited infarct size to 3.2 +/- 1.3% (N = 13, p < .0001). NKH477 (45 micrograms/kg) increased average cAMP levels in IP hearts during sustained ischemia to levels similar to those in untreated control hearts. However, NKH477 did not block IP (50.2 +/- 7.7% of the area at risk was infarcted in the control +NKH477 group [n = 10] versus 10.0 +/- 5.9% in the IP + NKH477 group [n = 7], P < .05). Therefore, we conclude that although IP lowers cAMP levels during sustained ischemia, this effect is not necessary for its protection against necrosis, since raising cAMP does not block this protection of IP.

Adenylyl Cyclases↗
PubMed1995

Phenotypic and molecular characterization of SerD, a dominant allele of the Drosophila gene Serrate.

The Drosophila gene Serrate (Ser) encodes a transmembrane protein with 14 epidermal growth factor--like repeats in its extracellular domain, which is required for the control of cell proliferation and pattern formation during wing development. Flies hetero- or homozygous for the dominant mutation SerD exhibit scalloping of the wing margin due to cell death during pupal stages. SerD is associated with an insertion of the transposable element Tirant in the 3' untranslated region of the gene, resulting in the truncation of the Ser RNA, thereby eliminating putative RNA degradation signals located further downstream. This leads to increased stability of Ser RNA and higher levels of Serrate protein. In wing discs of wild-type third instar larvae, the Serrate protein exhibits a complex expression pattern, including a strong stripe dorsal and a weaker stripe ventral to the prospective wing margin. Wing discs of SerD third instar larvae exhibit additional Serrate protein expression in the edge zone of the future wing margin, where it is normally not detectable. In these cells expression of wing margin specific genes, such as cut and wingless, is repressed. By using the yeast Gal4 system to induce locally restricted ectopic expression of Serrate in the edge zone of the prospective wing margin, we can reproduce all aspects of the SerD wing phenotype, that is, repression of wing margin-specific genes, scalloping of the wing margin and enhancement of the Notch haplo-insufficiency wing phenotype. This suggests that expression of the Serrate protein in the cells of the edge zone of the wing margin, where it is normally absent, interferes with the proper development of the margin.

Alleles↗
PubMed1994

Identification of an aprotinin antiviral domain.

Digestion of the proteinase inhibitor aprotinin, by clostripain, a cysteine proteinase, yielded five oligopeptide fragments. Two fragments exhibited both antiviral and antibacterial activities, two fragments only antiviral activity, and one fragment showed no antimicrobial activity. One of the former oligopeptides showed antiviral activity against human herpes simplex virus type 1 and bovine parainfluenza virus type 3. It consisted of the hexapeptide Y-F-Y-N-A-K corresponding to amino acids 21-26 of intact aprotinin. An identical synthetic peptide had the same antiviral spectrum as the natural hexapeptide, exhibited no antibacterial activity, and was also devoid of trypsin inhibiting activity. Intact aprotinin, in contrast, is ineffective against human herpes simplex virus 1 and bovine parainfluenza virus 3 but possesses antibacterial properties against several bacterial species [(1992) J. Appl. Bact. 72, 180-187].

Amino Acid Sequence↗
PubMed1994

The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation.

The Drosophila gene Serrate encodes a transmembrane protein with 14 EGF-like repeats in its extracellular domain. Here we show that loss-of-function mutations in this gene lead to larval lethality. Homozygous mutant larvae fail to differentiate the anterior spiracles, exhibit poorly developed mouth-hooks and show a severe reduction in the size of the wing and haltere primordia, which is not due to cell death. The few homozygous mutant escapers that pupariate develop into pharate adults that almost completely lack wings and halteres. Clonal analysis in the adult epidermis demonstrates a requirement for Serrate during wing and haltere development. Targeted ectopic expression of Serrate in the imaginal discs using the yeast transcriptional activator Gal4 results in regionally restricted induction of cell proliferation, e.g. the ventral tissues in the case of the wings and halteres. The results suggest that the wild-type function of Serrate is required for the control of position-specific cell proliferation during development of meso- and metathoracic dorsal discs, which in turn exerts a direct effect on morphogenesis.

Animals↗
PubMed1993

A new method for the insertion of multiple microprobes into neural and muscular tissue, including fiber electrodes, fine wires, needles and microsensors.

We developed a new method for the insertion of thin-shaft probes into neural and muscular tissue. Axial forces for driving the probes into tissue and radial forces against buckling are both provided by a stretched elastic rubber tube in which the probe is guided outside the tissue. Various geometric arrangements of arrays with independently advanceable probes are possible. Prototypes with 7 linearly aligned fiber electrodes and computer-controlled positioning motors were successfully used in single- and multiple-unit recordings from the visual system of awake monkeys (Eckhorn et al., 1993). The method is suitable in a wide range of applications, including insertion of fine microprobe fibers and wire electrodes into brain and muscle through the skin or dura, provided that the tips of the probes are sharp and hard enough.

Electric Wiring↗
PubMed1992

Bactericidal activities of lysozyme and aprotinin against gram-negative and gram-positive bacteria related to their basic character.

Bactericidal properties of aprotinin, a proteinase inhibitor and possibly a defence molecule in bovine species, and of chicken egg white lysozyme, known as muramidase, were investigated. Incubation of various bacteria in the presence of either aprotinin or lysozyme showed that both proteins killed Gram-positive as well as Gram-negative bacteria without addition of complement or EDTA. Denaturation of the two proteins by dithiothreitol did not lead to loss of their bactericidal potency. Electron microscopic examination of Escherichia coli incubated either with lysozyme or aprotinin revealed that the bacterial cytoplasms gradually disintegrated. Both aprotinin and lysozyme were demonstrated within the affected cytoplasm by immunogold labelling. The results suggest that the bactericidal potency of lysozyme is not only due to muramidase activity but also to its cationic and hydrophobic properties. The bactericidal activity of aprotinin is probably also related to both these properties rather than to its activity as proteinase inhibitor.

Animals↗