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M Spiess

Publications and source records attributed to M Spiess.

At least 37 records · Page 2Linked to original sources
PubMed1997

Experimental technique for (laparoscopic) bowel anastomosis: transient endoluminally stented anastomosis (TESA).

A new technique for bowel anastomosis is presented. The principle of transient endoluminally stented anastomosis (TESA) is based on anastomosing the two bowel ends around a resorbable stent of polyglycolic acid (PGA) in seroserosal contact. To evaluate the feasibility of TESA for bowel anastomosis, laparoscopic colon anastomosis following sigma resection was performed in five juvenile pigs. Three animals were sacrificed 2 months postoperatively, and the anastomoses were examined radiologically and histologically. One animal was sacrificed at day 2, suffering from acute peritonitis due to small bowel leak but with regular colon anastomosis. One trial was terminated at the fourth postoperative day because of insufficiency of the colon anastomosis. Three animals did not have any complications during the 2-month follow-up. In these animals the colon anastomoses were not detectable radiologically at the time of death. The microscopic examination showed intact mucosal and muscular layers without foreign material. Our study demonstrates that laparoscopic application of TESA to colon anastomosis is a feasible method. These results will further stimulate our future research for an anastomosis technique avoiding remnant foreign material.

Anastomosis, Surgical↗
PubMed1996

The oligomerization domain of the asialoglycoprotein receptor preferentially forms 2:2 heterotetramers in vitro.

The human hepatic asialoglycoprotein receptor is a noncovalent hetero-oligomer composed of two homologous subunits, H1 and H2, with an as yet unknown stoichiometry. Ligand specificity and binding affinity depend on the arrangement of the subunits in the complex. An 80-amino acid segment connecting the transmembrane and the carbohydrate binding domains contains heptad repeats characteristic of alpha-helical coiled coil structure. We expressed and purified corresponding peptides, H1S and H2S, and confirmed by circular dichroism spectroscopy that they can assume alpha-helical conformation. Oxidative cross-linking of amino-terminal cysteines generated specific covalent oligomers, indicating that separately H1S forms trimers and H2S tetramers. Upon mixing, covalent heterotetramers were formed with a preferred stoichiometry of 2 H1S and 2 H2S peptides. These results suggest that the stalk segments of the receptor subunits oligomerize to constitute an alpha-helical coiled coil stalk on top of which the carbohydrate binding domains are exposed for ligand binding. We propose that the functional asialoglycoprotein receptor is a 2:2 heterotetramer.

Amino Acid Sequence↗
PubMed1996

In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting.

To analyze the interaction of sorting signals with clathrin-associated adaptor complexes, we developed an in vitro assay based on surface plasmon resonance analysis. This method monitors the binding of purified adaptors to immobilized oligopeptides in real time and determines binding kinetics and affinities. A peptide corresponding to the cytoplasmic domain of wild-type influenza hemagglutinin, an apical membrane protein that is not endocytosed, did not significantly bind adaptor complexes. However, peptide sequences containing a tyrosine residue that has previously been shown to induce endocytosis and basolateral sorting were specifically recognized by adaptor complexes. The in vitro rates of adaptor association with these peptides correlated with the internalization rates of the corresponding hemagglutinin variants in vivo. Binding was observed both for purified AP-2 adaptors of the plasma membrane and for AP-1 adaptors of the Golgi, with similar apparent equilibrium dissociation constants in the range 10(-7)-10(-6) M. Adaptor binding was also demonstrated for a sequence containing a C-terminal di-leucine sequence, the second major motif of endocytosis/basolateral sorting signals. These results confirm the concept that interaction of cytoplasmic signals with plasma membrane adaptors determines the endocytosis rate of membrane proteins, and suggest the model that clathrin-coated vesicles of the trans-Golgi network are involved in basolateral sorting.

Adaptor Protein Complex alpha Subunits↗
PubMed1996

Morphological asymmetries of motoneurons innervating upper extremities: clues to the anatomical foundations of handedness?

Handedness is one of the main issues in laterality research and is known to be related to a large number of morphological asymmetries of the central nervous system. However, the main focus of previous studies were cerebral structures, which ignored the spinal cord as the most distal neural entity innervating the muscles of the extremities. We analyzed morphometrically motoneurons from segments innervating the arms and hands and compared them with motoneurons of segments that innervated the upper trunk. We found an asymmetry with larger motoneuron perikaryas on the right side of the spinal cord in segments innervating the upper limbs. To our knowledge this is the first time a morphological asymmetry on single-cell-level was shown in the spinal cord of man. The possible relation of this cellular asymmetry to the origins of handedness is discussed.

Aged↗
PubMed1995

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.

Amino Acid Sequence↗
PubMed1995

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Animals↗
PubMed1995

Heads or tails--what determines the orientation of proteins in the membrane.

The same translocation machinery in the endoplasmic reticulum translocates either the N-or the C-terminal domain of signal-anchor proteins across the membranes. Charged residues flanking the signal sequence are important to determine which end is translocated, but are not sufficient to generate a uniform topology. The folding state of the N-terminal segment, which is to be translocated posttranslationally, and the length or hydrophobicity of the signal sequence are additional criteria to determine protein orientation in the membrane.

Biological Transport↗
PubMed1995

High-affinity ligand binding to subunit H1 of the asialoglycoprotein receptor in the absence of subunit H2.

The hepatic asialoglycoprotein receptor is a hetero-oligomer composed of two homologous subunits. The specificity and affinity of ligand binding depends on the number and spatial arrangement of several galactose-binding sites within the receptor complex. Previous studies indicated that both subunits are required for high-affinity ligand binding, i.e. for the simultaneous interaction with three galactose residues within an N-linked glycan. However, we found that asialoorosomucoid (ASOR) and asialofetuin (ASF) bind to transfected COS-7 cells expressing subunit H1 in the absence of the second subunit H2. ASOR binding occurred with a dissociation constant of approximately 40 nM, approximately four-times higher than the Kd of ASOR binding to the hetero-oligomeric receptor. Normalized to the amount of H1 expressed, approximately 10-times fewer binding sites were produced by H1 alone. A glycopeptide with a single tri-antennary N-linked glycan purified from ASF bound to the hetero-oligomeric receptor, but did not bind detectably to H1-expressing COS-7 cells. H1 is thus unable to simultaneously recognize all three galactose residues in a glycan. From this, we conclude that, at a sufficiently high density of H1 on the cell surface, high-affinity binding of ASOR and ASF is the result of two or more glycans interacting with H1 oligomers with low affinity in a bivalent manner.

3T3 Cells↗
PubMed1995

New anastomosis technique for (laparoscopic) instrumental small-diameter anastomosis.

This study presents a new technique for visceral anastomosis. The principle consists of connecting the two parts to be anastomosed around a reabsorbable stent which is transluminally introduced into small-diameter viscus, where it is fixed. Advancing a larger tube along the axis of the machine, the larger, perforated viscus is inverted and pulled over the stent, and finally a rubber band pops off the machine endoluminally in order to fix the intestinal walls in seroserosal contact onto the stent. To evaluate this "micro" anastomosis, a biliary bypass (choledochojejunostomy and roux-en-y-loop) was performed in ten pigs. Nine of ten animals showed biliary bypass with good runoff in contrast radiography and completely reabsorbed stent after a 3-month follow-up. Weight gain, bilirubin, and alkaline phosphatase were normal. This technology demonstrates a safe and quick way to perform instrumental "micro" anastomosis without remnant foreign material.

Anastomosis, Roux-en-Y↗
PubMed1995

trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention.

Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40-amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and gamma-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain determines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s) for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins.

Amino Acid Sequence↗
PubMed1995

The asialoglycoprotein receptor is a potential liver-specific receptor for Marburg virus.

The liver is one of the main target organs of Marburg virus (MBG), a filovirus causing severe haemorrhagic fever with a high fatality rate in humans and non-human primates. MBG grown in certain cells does not contain neuraminic acid, but has terminal galactose on its surface glycoprotein. This observation indicated that the asialoglycoprotein receptor (ASGP-R) of hepatocytes may serve as a receptor for MBG in the liver. Binding studies revealed that the attachment of MBG to ASGP-R-expressing HepG2 cells, but not to ASGP-R-negative E6 Vero cells, has the characteristics of ligand binding to the ASGP-R: binding is dependent on calcium and is inhibited by excess asialofetuin and by anti-ASGP-R antiserum. Asialofetuin and the specific antiserum also inhibited MBG infection of HepG2 cells. In addition, it was shown that expression of ASGP-R cDNA in NIH 3T3 cells enhanced the susceptibility of these cells to MBG infection 4.5-fold. Interaction of MBG with the hepatic ASGP-R could thus explain the marked hepatotropism of the virus.

3T3 Cells↗
PubMed1994

A novel serine proteinase-like sequence from human brain.

From Alzheimer's visual cortex mRNA, a novel cDNA sequence with homology to the carboxy-terminal two-thirds of known serine proteinases was isolated (43% identity with human trypsinogen II). The corresponding mRNA has a size of approximately 8 kb and is expressed in low amounts in all human tissues tested.

Alzheimer Disease↗
PubMed1994

Tagging secretory and membrane proteins with a tyrosine sulfation site. Tyrosine sulfation precedes galactosylation and sialylation in COS-7 cells.

Sulfation of proteins on tyrosines is a late Golgi modification that can be used to label proteins with [35S]sulfate for the analysis of post-Golgi transport. To extend the use of this modification to proteins not naturally sulfated, we fused a tyrosine sulfation site, the carboxyl-terminal nonapeptide of cholecystokinin precursor, to the carboxyl terminus of two normally unsulfated proteins: alpha 1-proteinase inhibitor, a secretory protein, and subunit H1 of the asialoglycoprotein receptor; a type II membrane protein. The tagged proteins were efficiently sulfated in transfected COS-7 and Madin-Darby canine kidney cells. Specifically in COS-7 cells, the proteins were sulfated before they were galactosylated and sialylated and were converted to the mature forms with a half-time of approximately 2-3 min. This is in contrast to other cell types in which tyrosine sulfation was found to be virtually the last modification of the Golgi apparatus. Our results suggest that tyrosine sulfation occurs before the trans-Golgi in transfected COS-7 cells.

Amino Acid Sequence↗
PubMed1994

The two subunits of the asialoglycoprotein receptor contain different sorting information.

The human asialoglycoprotein receptor, an endocytic transport receptor of the basolateral surface of hepatocytes, is a hetero-oligomer of two homologous subunits H1 and H2. The cytoplasmic domain of H1 has been shown previously to contain a tyrosine-based signal for endocytosis and basolateral sorting. Here, we have investigated sorting determinants within subunit H2 and their contribution to the targeting of the hetero-oligomeric receptor complex. Despite extensive sequence homology, H2 expressed separately in fibroblast cells was endocytosed poorly, and mutation of phenylalanine 5 (corresponding to the critical tyrosine in H1) did not further reduce internalization. Consistent with this observation, ligand uptake by receptors composed of H1 lacking tyrosine 5 and H2 was inefficient. With respect to polarized transport in Madin-Darby canine kidney cells, H2 could not be analyzed separately, because in the absence of H1 subunit H2 was completely degraded intracellularly. Coexpression of both subunits yielded ligand-binding receptors located specifically on the basolateral surface. The mutant H1(5A) (tyrosine 5 replaced by alanine) is approximately 55% apical in the absence of H2. In cells expressing H1(5A) together with H2, however, subunit H2 directed receptor complexes exclusively to the basolateral domain. Phenylalanine 5 is not essential for basolateral transport. Thus, whereas the endocytosis signal of the hetero-oligomeric asialoglycoprotein receptor resides exclusively in subunit H1, polarized transport to the basolateral domain of Madin-Darby canine kidney cells may involve two signals, only one of which is active for endocytosis.

Amino Acid Sequence↗
PubMed1994

The multimodal water jet dissector--a technology for laparoscopic liver surgery.

UNLABELLED: In order to avoid laparotomy in patients with superficially and/or anatomically favourably located liver lesions, the feasibility of laparoscopic liver resection using an aqua jet was evaluated in 6 pigs. For this purpose a commercially available water jet dissector was adapted for laparoscopic use and modified to a multifunctional device providing aqua jet, suction and cautery. To improve laparoscopic vision during jet activity a hydrolaparoscope was used. The left lateral lobe was resected using clips for haemostasis only. RESULTS: All animals (34-55 kg, average 41 kg) survived the procedure. The blood loss at operation consisted of 225 ml (50-600 ml), the haematocrit 3.3% (1-8%). No bleeding or other complications were encountered postoperatively. The average weight of the specimens was 201 g (120-289 g). CONCLUSIONS: Laparoscopic liver resection using an aqua jet is feasible. Safe haemostasis can be achieved with clips if the vessels are entirely freed with the jet before clipping.

Animals↗