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Biomedical subjects

A Schweizer

Publications and source records attributed to A Schweizer.

At least 55 records · Page 3Linked to original sources
PubMed1998

Haemodynamic and metabolic changes induced by repeated episodes of hypoxia in pigs.

BACKGROUND: Repeated hypoxia and surgical trauma trigger a potent neuroendocrine response and their association is thought to play a pivotal role in the pathogenesis of multi-organ dysfunction. We investigated the cardiovascular and metabolic responses to repeated acute hypoxia in anaesthetised and surgically instrumented pigs. METHODS: Under ketamine-midazolam anaesthesia, 15 pigs were surgically instrumented for measurements of cardiac output, vascular pressures and organ blood flows. Lactate production and O2 uptake were determined in the brain, liver, kidney and intestine. Ten animals were subjected to two 12-min periods of ventilatory hypoxia (FIO2 = 7%) followed by re-oxygenation and 5 animals underwent 120-min normoxic ventilation (Control group). RESULTS: Both hypoxic challenges produced a comparable release of catecholamines that was associated with increased cardiac output and redistribution of blood flow away from the intestinal and renal areas towards the brain and the liver; O2 uptake was markedly reduced in the intestine (-56 +/- 10%, P < 0.05) and least affected in the brain and the kidney (-19 +/- 12% and -23 +/- 21%, respectively). During the second hypoxic test, lethal cardiovascular depression occurred in 5 animals; these non-survivors demonstrated impaired hyperdynamic response and incomplete recovery of intestinal O2 uptake during the first hypoxia/reoxygenation test. In the Control group, normoxic ventilation was not associated with significant haemodynamic and metabolic changes. CONCLUSION: Intraoperative hypoxia causes marked heterogeneity in organ blood flow and metabolism. The inability to develop a hyperdynamic cardiovascular response during a first hypoxic event, as well as a persistent intestinal O2 debt following re-oxygenation, predict the occurrence of death during the second hypoxic insult.

Anesthesia↗
PubMed1998

The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment.

To establish recycling routes in the early secretory pathway we have studied the recycling of the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 in HepG2 cells. Immunofluorescence microscopy showed progressive concentration of ERGIC-53 in the Golgi area at 15 degreesC. Upon rewarming to 37 degreesC ERGIC-53 redistributed into the cell periphery often via tubular processes that largely excluded anterograde transported albumin. Immunogold labeling of cells cultured at 37 degreesC revealed ERGIC-53 predominantly in characteristic beta-COP-positive tubulo-vesicular clusters both near the Golgi apparatus and in the cell periphery. Concentration of ERGIC-53 at 15 degreesC resulted from both accumulation of ERGIC-53 in the ERGIC and movement of ERGIC membranes closer to the Golgi apparatus. Upon rewarming to 37 degreesC the labeling of ERGIC-53 in the ERGIC rapidly returned to normal levels whereas ERGIC-53's labeling in the cis-Golgi was unchanged. Temperature manipulations had no effect on the average number of ERGIC-53 clusters. Density gradient centrifugation indicated that the surplus ERGIC-53 accumulating in the ERGIC at 15 degreesC was rapidly transported to the ER upon rewarming. These results suggest that the ERGIC is a dynamic membrane system composed of a constant average number of clusters and that the major recycling pathway of ERGIC-53 bypasses the Golgi apparatus.

Albumins↗
PubMed1997

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail.

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.

Amino Acid Sequence↗
PubMed1997

Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations.

Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.

Amino Acid Sequence↗
PubMed1996

Thermoregulatory and metabolic responses following cardiac surgery.

The thermoregulatory responses in 13 haemodynamically stable patients were evaluated over a 7-h period following coronary artery bypass graft surgery. Cutaneous vasoconstriction was defined by a forearm-to-fingertip skin temperature gradient (delta Tskin) > or = 4 degrees C and muscular hyperactivity was assessed by integrating the electromyographic signals (iEMG). Blood gases and lactate were measured in arterial, mixed venous and femoral venous blood. After arrival in ICU, we observed: (1) mild central hypothermia (Tc = 34.8 +/- 0.6 degrees C) and elevated peripheral vascular resistance in all the patients; (2) delta Tskin > or = 4 degrees C in 11 of 13 patients that persisted until a median Tc of 37.1 degrees C was achieved; (3) a transient increase in iEMG (+ 18%) was accompanied by an increase in total body O2 uptake (+ 28%); (4) elevated muscle O2 extraction rate (56 +/- 18% after 3 h) and lactate release. These data suggest that residual hypothermia in sedated patients after cardiac surgery elicits appropriate thermoregulatory responses that are associated with transient O2 supply/uptake mismatch within skeletal muscles.

Acid-Base Equilibrium↗
PubMed1996

The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane.

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.

Amino Acid Sequence↗
PubMed1996

Cysteine34 of the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting.

We have examined whether the two cysteine residues (Cys30 and Cys34) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-PAGE. Both Cys30 and Cys34 were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor. The palmitate rapidly turned over with a half-life of approximately 2 h compared to a half-life of greater than 40 h for the protein. Mutation of Cys34 to Ala resulted in the gradual accumulation of the receptor in dense lysosomes and the total loss of cathepsin D sorting function in the Golgi. A Cys30 to Ala mutation had no biologic consequences, showing the importance of Cys34. Mutation of amino acids 35-39 to alanines impaired palmitoylation of Cys30 and Cys34 and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sorting. These data suggest that palmitoylation of Cys30 and Cys34 leads to anchoring of this region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys34 is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor.

Amino Acid Sequence↗
PubMed1995

Evidence for a glycyl-proline transport system in ovine enterocyte brush-border membrane vesicles.

A peptide transport system in ovine enterocyte BBMV has been examined with glycyl-L-proline (Gly-Pro) as substrate. Transport is stimulated by an inwardly directed proton gradient (pH1 = 8.4; pH0 = 6.0). Gly-Pro uptake was saturable and conformed to Michaelis-Menten kinetics. Uptake was not affected by the presence of glycine but was inhibited by glycyl-L-phenylalanine (Gly-Phe). This is thought to be the first positive demonstration of the existence of a peptide transporter in the ruminant small intestine.

Animals↗
PubMed1995

Determination of the structural requirements for palmitoylation of p63.

Palmitoylation of p63, a type II membrane protein localized in the endoplasmic reticulum, is induced in a reversible manner by the drug brefeldin A. To study the requirements for palmitoylation, mutant forms of p63 were expressed in COS cells and analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis. By investigating deletion and point mutations, Cys100 in the 106-amino acid cytoplasmic tail of p63 has been identified as the site of acylation. Site-directed mutagenesis of residues 99-105 together with cytoplasmic tail truncation mutants showed that the amino acids surrounding Cys100 are not critical for palmitoylation of this residue. Analysis of a chimeric construct between p63 and the plasma membrane protein dipeptidylpeptidase IV further revealed that p63 palmitoylation is not dependent on its transmembrane domain. In contrast, the six-amino acid distance between the end of the predicted transmembrane domain and the palmitoylation site was found to be essential for proper acylation of p63.

Amino Acid Sequence↗
PubMed1995

Confirmed clinical efficacy of Actovegin in elderly patients with organic brain syndrome.

A double-blind randomized clinical trial was performed comparing the therapeutic effects of Actovegin versus placebo in elderly patients with organic brain syndrome. In addition to the necessary basic internal medicine therapy, 40 geriatric patients received dialy intravenous infusions of 250 ml Actovegin 20% p.i., and 20 patients received 250 ml 0.9% saline solution as placebo over a period of four weeks. Of the patient sample, 58% were hospitalized for simple dementia (ICD-9: 290.0) and 42% due to senile dementia with depressive or paranoid symptoms (ICD-9: 290.2). Based on the Syndrome Short Test (SKT) and the Sandoz Clinical Assessment Geriatric Scale (SCAG) score, the patients suffered from mild to moderate dementia. The therapeutic effect on the total SCAG score and the Clinical Global Impression (CGI) were the primary study variables. The scores for the SCAG subscales and the SKT score served as secondary variables. The mean total SCAG score in the drug group decreased from 56.3 at the start of therapy to 36.3 points at the end of therapy, and in the placebo group the total score went from 61.2 to 52.0 (p < 0.01). The CGI showed that with Actovegin, 70% of the patients experienced "distinct improvement" or "improvement" compared to only 35% with such results in the placebo group. The SCAG subscales and the total SKT score also demonstrated the superior effects of Actovegin compared to placebo. Moreover, the therapy group treated with Actovegin showed greater improvements in social behavior and mental performance than did the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes.

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.

Amino Acid Sequence↗
PubMed1995

Reassessment of the subcellular localization of p63.

p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.

Amino Acid Sequence↗
PubMed1994

Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment.

The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol. 113, 45-54) which provides a means to study more precisely the compartmentalization of the various enzymic functions along the early secretory pathway. Here, we have investigated if O-glycan initiation and sphingomyelin synthesis are associated with the ERGIC by analyzing both the responsible enzyme activities and their corresponding products. Moreover, the synthesis of glucosylceramide, the precursor of most glycosphingolipids, was also analyzed. In the purified ERGIC fraction UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) was only minimally enriched, sphingomyelin synthase was not enriched, and UDP-glucose:ceramide-glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, sphingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against GalNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with ERGIC-53. This was particularly evident in brefeldin A-treated cells which showed entirely different patterns of Tn antigens and ERGIC-53. The results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis mainly occur beyond the ERGIC in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers.

The type II membrane protein p63 is a resident protein of a membrane network interposed between rough ER and Golgi apparatus. To study the retention of p63, mutant forms were expressed in COS cells and the intracellular distribution determined by immunofluorescence microscopy. Investigation of chimeric constructs between p63 and the plasma membrane protein dipeptidylpeptidase IV showed that protein sequences from all three domains of the p63 protein are required to achieve complete intracellular retention. Mutational analysis of the 106-amino acid cytoplasmic tail of p63 revealed that the NH2-terminal 23 amino acids are necessary for retention. When p63 was solubilized with Triton X-100 and subjected to centrifugation at 100,000 g, it formed large, insoluble oligomers, particularly at neutral pH and below. A comparison of the behavior of wildtype and mutant p63 proteins in this assay revealed a perfect correlation between the formation of large oligomers and correct intracellular retention. These results suggest that self-association may be a major mechanism by which p63 is retained between the rough ER and the Golgi apparatus.

Amino Acid Sequence↗