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Biomedical subjects

A Girod

Publications and source records attributed to A Girod.

16 recordsLinked to original sources
PubMed1999

The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum.

To investigate the role of the KDEL receptor in the retrieval of protein toxins to the mammalian cell endoplasmic reticulum (ER), lysozyme variants containing AARL or KDEL C-terminal tags, or the human KDEL receptor, have been expressed in toxin-treated COS 7 and HeLa cells. Expression of the lysozyme variants and the KDEL receptor was confirmed by immunofluorescence. When such cells were challenged with diphtheria toxin (DT) or Escherichia coli Shiga-like toxin 1 (SLT-1), there was no observable difference in their sensitivities as compared to cells which did not express these exogenous proteins. By contrast, the cytotoxicity of Pseudomonas exotoxin A (PE) is reduced by expressing lysozyme-KDEL, which causes a redistribution of the KDEL receptor from the Golgi complex to the ER, and cells are sensitised to this toxin when they express additional KDEL receptors. These data suggest that, in contrast to SLT-1, PE can exploit the KDEL receptor in order to reach the ER lumen where it is believed that membrane transfer to the cytosol occurs. This contention was confirmed by microinjecting into Vero cells antibodies raised against the cytoplasmically exposed tail of the KDEL receptor. Immunofluorescence confirmed that these antibodies prevented the retrograde transport of the KDEL receptor from the Golgi complex to the ER, and this in turn reduced the cytotoxicity of PE, but not that of SLT-1, to these cells.

ADP Ribose Transferases↗
PubMed1998

A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry.

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.

Amides↗
PubMed1997

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND: Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS: The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS: The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of protein kinase Cdelta (PKCdelta) at threonine 505 is not a prerequisite for enzymatic activity. Expression of rat PKCdelta and an alanine 505 mutant in bacteria in a functional form.

A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes alpha, betaI, and betaII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative "PKC kinase" for permissive activation. Expression of PKCalpha in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCalpha and PKCbetaII could be identified as Thr497 and Thr500, respectively. We report here that PKCdelta, contrary to PKCalpha, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCdelta expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCalpha and PKCbetaII, respectively, is not essential for obtaining a catalytically competent conformation of PKCdelta. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCdelta and that at least this PKC isoenzyme differs from the isotypes alpha, betaI, and betaII regarding the permissive activation by a PKC kinase.

Alanine↗
PubMed1996

Crystal structures of catalytic subunit of cAMP-dependent protein kinase in complex with isoquinolinesulfonyl protein kinase inhibitors H7, H8, and H89. Structural implications for selectivity.

The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 A) and with H89 (2.3 A) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

In vivo activation of recombinant cAPK catalytic subunit active site mutants by coexpression of the wild-type enzyme, evidence for intermolecular cotranslational phosphorylation.

The catalytic subunit of cAMP dependent protein kinase (cAPK) carries two stable autophosphorylated residues. One of them, Thr197, resides in the so-called protein kinase activation segment, and needs to be phosphorylated for full activity and protein kinase inhibitor binding of the enzyme. While wild-type recombinant mammalian C-subunit, expressed in E. coli, can fully autoactivate itself by phosphorylation at Thr197, many active site mutants lack this autophosphorylation activity, so that the primary effects of the mutations become obscured. Two active site mutants of bovine C-subunit, defective in protein kinase inhibitor peptide binding, were activated by wild-type enzyme in vivo, but could not be activated in vitro, demonstrating intermolecular and presumably cotranslational autophosphorylation. The results may delineate strategies for the expression and mutagenesis of other protein kinases with requirements for activation segment phosphorylation.

Amino Acid Sequence↗
PubMed1996

Homologous and nonhomologous retroviral recombinations are both involved in the transfer by infectious particles of defective avian leukosis virus-derived transcomplementing genomes.

We previously described avian leukosis virus-based packaging cell lines that produce stocks of retroviral vectors in which replication-competent viruses were not detectable. However, following infection of target cells with these retroviral stocks, we recently obtained colonies resulting from the transmission of recombinant genomes. Here, we have analyzed their genetic structure and shown that (i) each of them results from recombination between the packaging- and integration-defective transcomplementing genomes and the retroviral vector; (ii) recombination probably occurred during the reverse transcription step, involving strand switching of the reverse transcription growing point from the infectious retroviral vector to the transcomplementing RNA; and (iii) sequence identity and nonhomologous sequences were both used for the strand switching.

Animals↗
PubMed1995

Analysis of ALV-based packaging cell lines for production of contaminant defective viruses.

We have previously described avian leukosis virus-based packaging cell lines that express gag, pol, and env proteins from two transcomplementing genomes and produce helper-free stocks of retroviral vectors with different host ranges. In this report, we demonstrated that (i) despite the deletion of the psi packaging sequence, the packaging-defective transcomplementing retroviral transcripts were packaged into virions at a level that could reach 2.3% of a wild-type virus packaging level and (ii) despite deletion of the 3' LTR, these genomes were transferred along with the vector to target cells. As these genomes were also bearing a selectable gene, titers of the resulting contaminant particles could be estimated, depending on the cell line to be between 0 and 6 infectious particles/ml of supernatant.

Animals↗
PubMed1995

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.

Animals↗
PubMed1993

Use of helper cells with two host ranges to generate high-titer retroviral vectors.

We have recently described Avian Leukosis Virus (ALV)-based packaging cell lines that can produce helper-free ALV-based retrovirus vectors with A, B, C, and E envelope host ranges. Here, we report that lacZ retroviral vectors of subgroup C or E can infect helper cells of subgroup A (Isolde) which are then able to produce high titers of lacZ recombinant viruses of subgroup A. Superinfection of helper cells by lacZ recombinant virus was performed by cocultivating packaging cells with two subgroup specificities (A and E), but this did not result in increased recombinant virus titers. This "ping-pong" process caused the emergence of replication-competent (RC) viruses which are shown to result from recombination between the viral sequences of the helper cell lines and the cis-acting sequences of the lacZ recombinant virus.

Avian Leukosis Virus↗
PubMed1990

[Two-years' experience of ambulatory anesthesia in otorhinolaryngology in children].

Three hundred children were anaesthetized for E.N.T. operations. The anaesthetic consultation took place one week prior to operation. The anaesthetic comprises premedication, inhalation of halothane for infants, or propofol drip for the others with tracheal tubing for 10% of them. Recovery in every case is supervised in the recovery room. There are few setbacks with this method. Eleven children were not accepted for operation for sociological or medical reasons (Willebrand diseases) and only 3 had to remain in hospital until the following day due to slow recovery from anaesthetic.

Adenoidectomy↗
PubMed1987

[Use of propofol in ophthalmological anesthesia in elderly patients].

Forty patients more than 65 years old were anaesthetized with propofol for cataract surgery. Patients were divided into two groups: those intubated after topical oropharyngeal anaesthesia and those intubated after muscle relaxation obtained with suxamethonium. Induction and maintenance of anaesthesia were carried out with 2.5 mg . kg-1 and 4 mg . kg-1 . h-1 respectively. Concurrent use of muscle relaxants had no effect on the action and characteristics of the general anaesthesia or recovery. Relative bradycardia (7%) and arterial hypotension (25%) occurred rapidly following induction and continued during the entire period of anaesthesia despite the systematic infusion of 500 ml of electrolyte solutions. Recovery was extremely rapid. The original Glasgow score was obtained within 20 min. The only negative side-effects were generally late occurring (4-6 h): postanaesthetic nausea or vomiting. Otherwise, tolerance was excellent. Use of propofol was ideal for this type of surgery due to its strong and durable ocular hypotensive effect.

Aged↗