Biomedical subjects

L Muller

Publications and source records attributed to L Muller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Insertion time of the pulmonary artery catheter in critically ill patients.

OBJECTIVES: Measurement of the time elapsed from the decision to use a pulmonary artery catheter to the onset of the adapted treatment. DESIGN: Prospective study. SETTING: Critical care unit of a university hospital. PATIENTS: A total of 104 critically ill patients. INTERVENTIONS: The time elapsed from the decision to use a pulmonary artery catheter to the onset of the adapted treatment. Five time intervals (availability, preparation, catheterization, data collection, and therapeutic intervals) were individualized according to the times of decision of pulmonary artery catheter insertion, operator's hand washing, venipuncture, postoperative dressing, data collection, and the effective onset of subsequent therapy. MEASUREMENTS AND MAIN RESULTS: Among 120 used pulmonary artery catheters, seven could not be inserted. The time to use the pulmonary artery catheter was never shorter than 45 mins (median value = 120 mins). For availability, preparation, catheterization, data collection, and therapeutic intervals, the median values were 30, 20, 20, 20, and 10 mins, respectively. The availability and data collection intervals were shortened during the night period and the fourth quarter of the study, respectively. CONCLUSIONS: The pulmonary artery catheter use is time consuming. However, the availability and data collection intervals could be shortened.

Adult↗

PubMed2000

Expression of the endothelin-converting enzyme-1 isoforms in endothelial cells.

The transformed human endothelial cell line EA.hy926 is commonly used for studying in vitro different aspects of endothelial cell biology such as signal transduction, expression or angiogenesis. These cells have the ability to process big endothelin (big-ET) into endothelin (ET), and express the endothelin-converting enzyme ECE-1. Several isoforms of ECE-1 which differ only in their N-terminal part (i.e. the end of the cytosolic domain) have now been identified. We could detect the co-expression of all four isoforms. Recent works have shown that the variable cytosolic domain is responsible for the differential intracellular localization of ECE-1 isoforms. Using antibodies directed against ECE-1a and ECE-1b/c/d, we have characterized the intracellular distribution of these isoforms in EA.hy926 cells by immunofluorescence. Electron microscopy allowed us to identify further the intracellular compartment that contains ECE-1 as multivesicular bodies, a compartment involved in the endocytic pathway. In addition, using an antibody directed against the catalytic domain, we could demonstrate that no monomeric ECE-1 is present at the plasma membrane. Indeed, detection of ECE-1 immunoreactivity at the cell surface of living cells required a dithiothreitol (DTT) treatment. Altogether, these results demonstrate that the EA.hy926 cell line is a helpful model for studying the regulation of the production of endothelin by ECE.

Animals↗

PubMed2000

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Affinity Labels↗

PubMed1999

A 36-residue peptide contains all of the information required for 7B2-mediated activation of prohormone convertase 2.

The prohormone convertases (PCs) are serine proteinases responsible for the processing of secretory protein precursors. PC2 is the only member of this family whose activation requires intracellular interaction with a helper protein, the neuroendocrine protein 7B2. In order to gain a better understanding of the mechanism of proPC2 activation, we have characterized the structural determinants of 7B2 required for proPC2 activation. We had already identified a proline-rich binding determinant in the 21-kDa domain, the portion of 7B2 responsible for proPC2 activation. We have now investigated the function of the weakly conserved amino-terminal portion of 21-kDa 7B2 by sequential deletions. Mutant proteins were analyzed in four assays: binding to proPC2, facilitation of proPC2 maturation, and activation of proPC2 in vivo and in vitro. We found that the amino-terminal half of 7B2 is not involved in proPC2 activation, and we identified an active 36-residue peptide that contains the previously characterized proline-rich sequence as well as an alpha-helix and the only disulfide bond of 7B2. Mutation of the alpha-helix and of the cysteines demonstrated that these determinants are absolutely required for PC2 activation. Thus, the 186-residue full-length 7B2 rat protein can be functionally reduced to an internal segment of only 36 residues.

Amino Acid Sequence↗

PubMed1999

The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease.

The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion.

Adipose Tissue↗

PubMed1999

The cell biology of the prohormone convertases PC1 and PC2.

Mature peptide hormones and neuropeptides are typically synthesized from much larger precursors and require several posttranslational processing steps--including proteolytic cleavage--for the formation of the bioactive species. The subtilisin-related proteolytic enzymes that accomplish neuroendocrine-specific cleavages are known as prohormone convertases 1 and 2 (PC1 and PC2). The cell biology of these proteases within the regulated secretory pathway of neuroendocrine cells is complex, and they are themselves initially synthesized as inactive precursor molecules. ProPC1 propeptide cleavage occurs rapidly in the endoplasmic reticulum, yet its major site of action on prohormones takes place later in the secretory pathway. PC1 undergoes an interesting carboxyl terminal processing event whose function appears to be to activate the enzyme. ProPC2, on the other hand, exhibits comparatively long initial folding times and exits the endoplasmic reticulum without propeptide cleavage, in association with the neuroendocrine-specific protein 7B2. Once the proPC2/7B2 complex arrives at the trans-Golgi network, 7B2 is internally cleaved into two domains, the 21-kDa fragment and a carboxy-terminal 31 residue peptide. PC2 propeptide removal occurs in the maturing secretory granule, most likely through autocatalysis, and 7B2 association does not appear to be directly required for this cleavage event. However, if proPC2 has not encountered 7B2 intracellularly, it cannot generate a catalytically active mature species. The molecular mechanism behind the intriguing intracellular association of 7B2 and proPC2 is still unknown, but may involve conformational rearrangement or stabilization of a proPC2 conformer mediated by a 36-residue internal segment of 21-kDa 7B2.

Animals↗

PubMed1999

Ammoniated forage poisoning: acute toxicity of newly identified dialkylimidazoles to inbred mice.

The i.p. acute toxicity of the dialkylimidazoles 1,2-dimethylimidazole, 1,4-dimethylimidazole, 1,5-dimethylimidazole, 2,4-dimethylimidazole and 2-ethyl-4-methylimidazole was investigated in C57B1 female mice, and compared to the toxicity of 4-methylimidazole (4-MI). An approximate median lethal dose protocol was applied designed to obtain a qualitative description of the clinical toxic effects and an estimate of the acute lethal dose with the use of a minimal number of animals. The approximate median lethal dose of the dialkylimidazoles varied from slightly above to 3-times greater than that of 4-MI. 1,4-Dimethylimidazole and 1,5-dimethylimidazole had tremorogenic and convulsive effects qualitatively similar to those of 4-MI. Testing a mixture of alkylimidazoles comparable to the composition found in toxic ammoniated forage or milk did not indicate any synergistic enhancement of the toxicity to mice.

Animal Feed↗

PubMed1999

[Comparation of the antero-lateral and posterior approaches in primary total hip arthroplasty].

Comparison between the antero-lateral and posterior approaches in primary total hip arthroplasty. In this retrospective study, 184 patients were enrolled, 95 submitted to the anterolateral (Watson jones) and 89 to the posterior approach (Moore) from June 1993 to June 1997. The outcomes assessed were perioperative data (operative time, hospital stay, time from surgery until hospital discharge, surgical bleeding and the need for blood transfusion), as well as late complications (deep venous thrombosis DVP, pulmonary embolism, periopheral nerve injury, prothesis instability and others). Both groups did not differ in terms of preoperative parameters. Those submitted to the posterior approach had shorter operative times (p < 0.001), as well as reduced bleeding (p < 0.05) and need for blood transfusion (p < 0.001) during surgery. The outcomes, such as late complications, had similar reduced frequency in both groups. The posterior approach has been successfully applied in our service and proves to be an excellent alternative surgical access to the total hip arthroplasty.

Adult↗

PubMed1998

Structural elements of PC2 required for interaction with its helper protein 7B2.

The structures of the eukaryotic subtilisin protease family members can be divided into four distinct domains as follows: the proregion, the catalytic domain, the P domain, and the carboxyl-terminal region. Although these enzymes are evolutionarily related, only prohormone convertase 2 (PC2) requires 7B2 for activation. To examine the potential contribution of each domain of PC2 to PC2-7B2 interactions, we performed sequential deletions, site-directed mutagenesis, and domain swapping to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments/amino acids of pro-PC1. These chimeras and mutant enzyme molecules were then expressed in AtT-20 cells and analyzed for 7B2 binding, maturation ability, and enzymatic activity. The results revealed that 1) the PC2 proregion is required but is not sufficient to confer 7B2 binding; 2) the P domain is required for the stabilization of PC2 structure and is not exchangeable with the P domain of PC1; and 3) the carboxyl-terminal domain is not involved in 7B2 binding. Site-directed mutagenesis of pro-PC2 further showed that a single residue replacement in the catalytic domain, Tyr-194 --> Asp, prevented pro-PC2 from binding 7B2 and blocked activation. This residue is present within a loop rich in aromatic amino acids which appears to be on the surface of the molecule as extrapolated from the crystal structure of subtilisin. This loop may represent the primary recognition site for 7B2 within the catalytic domain.

Amino Acid Sequence↗

PubMed1998

[Postoperative locoregional analgesia in the adult: epidural and peripheral techniques. Indications, adverse effects and monitoring].

Regional analgesia is a very effective way to treat postoperative pain. Lumbar and thoracic epidural analgesia are well adapted to major abdominal and thoracic surgery. Nevertheless, respiratory side effects induced by opioids are potentially severe and an adequate monitoring is essential. In orthopaedic surgery, perineural blocks are the best technique to manage postoperative pain and perineural catheters may be used. The importance of intra-articular analgesia, simple and safe, is not fully understood. The association of a local anaesthetic inducing a minor motor block and a strong sensitive block (bupivacaine, ropivacaine), with an opioid seems to be the best pharmacologic choice regarding quality of analgesia and safety. Benefits of postoperative regional analgesia on mortality and morbidity are not demonstrated. Medical and nursing staff and specialized units should improve quality of postoperative regional analgesia as well. General guidelines for the practice of regional anaesthesia must be closely followed.

Analgesia, Epidural↗

PubMed1998

Cloning and functional analysis of C. elegans 7B2.

The neuroendocrine protein 7B2 is a binding protein for the prohormone convertase 2 (PC2) and is required for the intracellular conversion of proPC2 to active PC2. Both full-length 7B2 and its carboxy-terminal 31-residue peptide (CT peptide) are capable of potent inhibition of PC2; the 7B2 protein thus regulates both the biosynthesis and the activity of PC2. Vertebrate 7B2s are highly conserved (92%-97% homology), and thus, species comparison has not been informative in assessing the crucial protein domains responsible for bioactivity. We here report the cloning of the Caenorhabditis elegans 7B2 protein. Although weakly conserved with the vertebrate sequences (23% similarity with mouse 7B2), C. elegans 7B2 contains the signature PPNPCP motif as well as a highly conserved heptapeptide within the CT peptide. In in vitro assays, C. elegans 7B2 possessed significant inhibitory activity against recombinant vertebrate PC2 (IC50 130 nM), and in two functional tests, the amino-terminal domain of C. elegans 7B2 facilitated the activation of proPC2. We conclude that despite low amino acid conservation overall, both functional domains within 7B2 have been conserved between the C. elegans and the vertebrate proteins.

Amino Acid Sequence↗

PubMed1998

Immunocytochemical localization of the prohormone convertases PC1 and PC2 in rat prolactin cells.

The prohormone convertases PC1 and PC2 are subtilisin-related endopeptidases that process prohormone and neuropeptide precursors. Using different ultrastructural immunocytochemical approaches, we have investigated their intracellular distribution in a neuroendocrine cell type that has not been examined thus far, the rat anterior pituitary lactotrope. These cells secrete mainly prolactin and also express the neuroendocrine-specific protein secretogranin II, which is considered a peptide precursor. Our study provides evidence for the expression of PC1 and PC2 in rat lactotropes and provides new information on their subcellular localization. Apart from their presence in the secretory granules, PC1 and PC2 displayed different major localization along the secretory pathway. PC1 immunoreactivity was concentrated in the Golgi apparatus, whereas PC2 immunoreactivity was prominent in the rough endoplasmic reticulum (RER). These observations provide morphological support for previous biochemical analysis of proPC1 and proPC2 post-translational processing, which has demonstrated that PC1 exits very rapidly from the RER, whereas PC2 is retained much longer in this compartment. (J Histochem Cytochem 46:101-108, 1998)

Animals↗

PubMed1997

Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding.

Among the members of the prohormone convertase (PC) family, PC2 has a unique maturation pattern: it is retained in the ER for a comparatively long time and its propeptide is cleaved in the TGN/ secretory granules rather than in the ER. It is also unique by its association with the neuroendocrine protein 7B2. This interaction results in the facilitation of proPC2 maturation and in the production of activatable proPC2 from CHO cells. In the present study, we have investigated the mechanism of this interaction. ProPC2 binds 7B2 in the ER, but exits this compartment much more slowly than 7B2. We found that proPC2 was also slow to acquire the capacity to bind 7B2, whereas 7B2 could bind proPC2 rapidly after synthesis. This indicated that proPC2 folding was the limiting step in the formation of the complex. Indeed, sensitivity of native proPC2 to N-glycanase F digestion and inhibition of proPC2 folding supported the notion that 7B2 is not involved in the early steps of proPC2 folding, and that proPC2 must fold before binding 7B2. Under experimental conditions that prevent propeptide cleavage, 7B2 expression increased proPC2 transport to the Golgi. This increase exhibited the same kinetics as the facilitation of the removal of the propeptide. Finally, proPC2 activation could be reconstituted in Golgi- enriched subcellular fractions. In vitro, 7B2 was required for proPC2 activation at an acidic pH. Taken together, our results demonstrate that rather than promoting proPC2 folding, 7B2 acts as a helper protein involved in proPC2 transport and is required in the proPC2 activation process. We propose, therefore, that 7B2 stabilizes proPC2 in a conformation already competent for these two events.

Animals↗

PubMed1997

Proteolytic processing of sulfated secretogranin II in the trans-Golgi network of GH3B6 prolactin cells.

Secretogranin II (SgII) is a protein specific to the matrix of the secretory granules in neurons and neuroendocrine cells. We have already demonstrated the precursor-product relationship between sulfated SgII and four N-terminal derived peptides in GH3B6 prolactin cells. In this study, we have investigated the subcellular compartment in which the cleavage of SgII is initiated by taking advantage of its tyrosine sulfation in the trans-Golgi network (TGN). In order to prevent export of radiosulfated SgII from the TGN, we used brefeldin A (BFA) as well as incubation at 20 degrees C. BFA completely inhibited the cleavage of SgII when added immediately post-pulse. BFA added a few minutes post-pulse or after a 20 degrees C incubation, however, permitted the cleavage of SgII in the presence of the drug. These SgII-derived peptides generated in the presence of BFA could not be released upon stimulation of the cells by either thyroliberin, a physiological secretagogue, or KCl. These results demonstrate that SgII can be cleaved in the TGN. They also evidence that the cleavage occurs in a distal compartment of the TGN different from the sulfation site. The transfer of SgII from the sulfation site to this distal compartment of the TGN involves BFA-sensitive membrane dynamics.

Animals↗

PubMed1995

Production and secretion of N-terminal secretogranin II derived peptides in GH3B6 prolactin cells.

Chromo/secreto-granins are proteins specific of neuroendocrine cells. Chromogranin B (CgB) and secretogranin II (SgII) are both present in normal and in tumoral (GH3B6) prolactin cells, in which they are colocalized in the same secretory granules. These proteins contain multiple dibasic cleavage sites and are considered as potential precursors of active peptides, though their exact function remains unknown. SgII is sulfated on tyrosine-126. We took advantage of this feature to study its post-translational processing in anterior pituitary cells in primary culture and in GH3B6 cells. Pulse-chase experiments with [35S]sulfate demonstrated the precursor-product relationship between SgII and four N-terminal-derived peptides. Kinetic experiments showed the sequential cleavage of SgII from the C-terminus to the N-terminus. Mature SgII and the derived peptides were secreted by both cell models, and their release was stimulated by TRH (30 nM), a secretagogue of prolactin. These data show that SgII is proteolytically processed into different secreted peptides in prolactin cells and demonstrate that GH3B6 cells provide a good cell model for studying the maturation of SgII in anterior pituitary.

Animals↗

PubMed1995

The outcome of babies of mothers with severe rhesus incompatibility treated at Tygerberg Hospital, 1980-1993.

OBJECTIVE: To determine the outcome of babies of mothers with severe rhesus (Rh) incompatibility treated by elective delivery when the amniotic optical density at 450 nm crossed Whitfield's action line (group 1), by plasmapheresis and immunotherapy (group 2) or by means of intra-uterine intravascular transfusions (group 3). STUDY DESIGN: A retrospective study of 55 mothers and their 57 fetuses with severe Rh incompatibility at < 34 weeks' pregnancy duration. MAIN OUTCOME PARAMETERS: Number of mothers in each treatment group, prevalence of intra-uterine death, hydrops, intra-uterine intravascular transfusions, cord haematocrit, cord bilirubin, number of liveborn babies, birth weight, neonatal death, hyaline membrane disease (HMD) and exchange transfusions. STUDY POPULATION AND SETTING: All mothers and babies with severe Rh incompatibility (defined as an amniotic optical density of 450 nm in the upper and upper-mid zone on the Liley chart at < 34 weeks' pregnancy duration, previous fetal hydrops or Rh-related intra-uterine death (IUD), fetal hydrops on ultrasound or a fetal haematocrit < 30% at cordocentesis) treated at Tygerberg Hospital between January 1980 and January 1993. There were 20 fetuses each in groups 1 and 3, and 17 in group 2. RESULTS: A total of 48 babies (84%) were liveborn and of these 74% survived the neonatal period. (ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Identification of multiple subunits of heterotrimeric G proteins on the membrane of secretory granules in rat prolactin anterior pituitary cells.

The subcellular distribution of multiple subunits of heterotrimeric GTP-binding proteins has been investigated in rat anterior pituitary cells in primary culture, and more precisely in prolactin cells, by immunocytochemistry and subcellular fractionation followed by immunoblotting or ADP ribosylation, using polyclonal affinity-purified antibodies directed against Gi3 alpha, Gs alpha, Go1 alpha, Go2 alpha, and G beta. As expected, all these subunits were detected on the plasma membrane. They were, however, also detected on the membrane of several intracellular compartments involved in the secretory pathway, particularly on the secretory granule membrane. Differences appeared between the precise subcellular distribution and the local concentration of each subunit. The main subunits present on the secretory granule membrane were Gi3 alpha and Gs alpha. Go1 alpha, Go2 alpha, and G beta were detected, to a lesser extent, on parts of the membrane of a few secretory granules located near the plasma membrane. Domains of the rough endoplasmic reticulum cisternae were immunolabeled with anti-Gs alpha and anti-Go1 alpha. In the Golgi zone, the membrane of some vesicles was stained only with anti-Gs alpha and anti-Go2 alpha. The association of this set of heterotrimeric G protein subunits on the membrane of the secretory granules suggests that these subunits could be involved in the regulation of formation, storage, targeting, and/or exocytosis of these organelles.

Animals↗

PubMed1994

[Block of the sciatic nerve].

Sciatic nerve blocks were seldom used until recently. They apply to most surgical procedures on the lower limb and are often combined with a "3 in 1" block. However they can be used alone for foot surgery or postoperative analgesia. Nerve stimulators make their realization easier and more reliable. Sciatic nerve block can be obtained by different techniques. The choice of which being helped by some guidelines according to the patients characteristics and the surgical site.

Anesthetics, Local↗