Biomedical subjects

M Aebi

Publications and source records attributed to M Aebi.

At least 109 records · Page 6Linked to original sources

PubMed1995

STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.

Amino Acid Sequence↗

PubMed1995

PCR-mediated cloning and sequencing of the DmOST50 gene, a WBP1/AvOST50/OST48 homologue, from Drosophila melanogaster.

Oligodeoxyribonucleotides were used in a PCR reaction to amplify the conserved region of the DmOST50 cDNA encoding an oligosaccharyltransferase subunit from Drosophila melanogaster (Dm). The amplified fragment was cloned and sequenced, and was then used as a homologous probe to isolate a DmOST50 cDNA from a lambda ZAP library. The deduced amino acid (aa) sequence of DmOst50p shows 27.1% identity with the corresponding sequence of the yeast Wbp1p, 62.4% identity with the avian AvOst50p and 62.7% with the canine Ost48p sequences. 17% of all aa residues were found to be identical among all species tested, indicating a high degree of conservation during evolution.

Amino Acid Sequence↗

PubMed1994

Site-directed mutagenesis of the yeast PRP20/SRM1 gene reveals distinct activity domains in the protein product.

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.

Alleles↗

PubMed1994

Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae.

UDP-glucose:dolichyl-phosphate glucosyltransferase is a transmembrane-bound enzyme of the endoplasmic reticulum involved in protein N-linked glycosylation. This enzyme catalyzes the transfer of glucose from UDP-glucose to dolichyl phosphate. The structural gene encoding this transferase from Saccharomyces cerevisiae was isolated by complementation of an alg5-1 mutation. DNA sequencing of ALG5 revealed an open-reading frame of 1002 bases encoding a transmembrane protein of molecular mass 38.3 kDa. Overexpression of Alg5p in both yeast and Escherichia coli results in an increase of UDP-glucose:dolichyl-phosphate glucosyltransferase activity, whereas a deletion of the yeast gene leads to a loss of this activity and a concomitant underglycosylation of carboxypeptidase Y. The ALG5 protein has sequence similarity to the GDP-mannose:dolichyl-phosphate mannosyltransferase (Dpm1p) from S. cerevisiae. Topological studies indicate that UDP-glucose:dolichyl-phosphate glucosyltransferase is a transmembrane protein that spans the membrane several times.

Amino Acid Sequence↗

PubMed1994

New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus.

Glc3Man9GlcNAc2 is the preferred substrate of the oligosaccharyltransferase of N-linked glycosylation of proteins, but nonglucosylated oligosaccharides can be transferred to proteins in Saccharomyces cerevisiae. Mutations affecting the addition of the three terminal glucose residues lead to accumulation of Man9GlcNAc2 or Glc1Man9GlcNAc2 in vivo but do not show any detectable growth defect. When these mutations were introduced into a strain with reduced oligosaccharyltransferase activity (due to the wbp1-1 mutation), a severe growth defect was observed: accumulation of suboptimal lipid-linked oligosaccharide and reduced oligosaccharyltransferase activity resulted in a severe underglycosylation of secreted proteins. This new synthetic phenotype made it possible to isolate the ALG8 locus, encoding a potential glucosyltransferase of the endoplasmic reticulum. The ALG8 protein is a 63.5-kDa hydrophobic protein that is not essential for the vegetative growth of yeast. However, the lack of this protein resulted in underglycosylation of secreted proteins.

Amino Acid Sequence↗

PubMed1994

The genetic interaction of kar2 and wbp1 mutations. Distinct functions of binding protein BiP and N-linked glycosylation in the processing pathway of secreted proteins in Saccharomyces cerevisiae.

The endoplasmic binding protein BiP and N-linked glycosylation are proposed to be essential components in the processing pathway of secreted protein. In Saccharomyces cerevisiae, BiP is encoded by the KAR2 gene; WBP1 encodes an essential component of the N-oligosaccharyltransferase complex. wbp1 mutations result in reduced oligosaccharyltransferase activity and a temperature-sensitive phenotype. We show that a combination of kar2 and wbp1 mutations results in a synthetic phenotype with a strongly reduced growth rate at the permissive temperature. To investigate the role of N-linked glycosylation in BiP function, the processing of non-glycosylated carboxypeptidase was followed in different kar2 strains at the permissive temperature. In all kar2 strains, the processing of non-glycosylated carboxypeptidase Y was drastically reduced. A specific BiP/non-glycosylated carboxypeptidase Y complex was detected in kar2-159 and kar2-203 cells whereas the kar2-1 mutation did not result in such a complex. Our data show that BiP and N-linked glycosylation are directly involved in the processing of secreted proteins. The results support the hypothesis that BiP stabilizes the folding-competent and assembly-competent state of a polypeptide, whereas N-linked oligosaccharides are structural components required in the folding process after the polypeptide is released from BiP.

Endoplasmic Reticulum↗

PubMed1994

Cervical discography in discogenic pain syndrome and its predictive value for cervical fusion.

We have reviewed 27 patients who underwent fusion of a total of 39 cervical levels for discogenic pain syndrome. All patients were diagnosed by positive discography defined as provocation of their characteristic pain on a Adams type 3-5 contrast distribution pattern. The mean age of the patients was 46.4 years and the follow-up averaged 16.2 months (range 9-42 months). Fusion was performed through a ventral approach with the use of an iliac bone graft in all patients. There were 22 one-level, 7 two-level and 1 three-level fusions. Overall 19 patients (73%) had good to excellent results according to the criteria of Simmons and Segil. Six patients (23%) had a fair outcome and one (3.8%) a poor result. More good to excellent results (85.7%) were seen after two-level fusions than after one-level fusions (61.9%). Patients presenting with pain radiation to the upper limbs had a more favorable outcome. Better results also were found in patients with pain onset after cervical spine trauma than those without. However, due to the limited number of patients in this group, differences did not reach statistically significant levels. Reported postoperative results in the literature in similar patient groups without preoperative evaluation of the symptomatic level by discography show a good to excellent outcome in a less favorable range of 35%-46%.

Adult↗

PubMed1994

A comprehensive classification of thoracic and lumbar injuries.

In view of the current level of knowledge and the numerous treatment possibilities, none of the existing classification systems of thoracic and lumbar injuries is completely satisfactory. As a result of more than a decade of consideration of the subject matter and a review of 1445 consecutive thoracolumbar injuries, a comprehensive classification of thoracic and lumbar injuries is proposed. The classification is primarily based on pathomorphological criteria. Categories are established according to the main mechanism of injury, pathomorphological uniformity, and in consideration of prognostic aspects regarding healing potential. The classification reflects a progressive scale of morphological damage by which the degree of instability is determined. The severity of the injury in terms of instability is expressed by its ranking within the classification system. A simple grid, the 3-3-3 scheme of the AO fracture classification, was used in grouping the injuries. This grid consists of three types: A, B, and C. Every type has three groups, each of which contains three subgroups with specifications. The types have a fundamental injury pattern which is determined by the three most important mechanisms acting on the spine: compression, distraction, and axial torque. Type A (vertebral body compression) focuses on injury patterns of the vertebral body. Type B injuries (anterior and posterior element injuries with distraction) are characterized by transverse disruption either anteriorly or posteriorly. Type C lesions (anterior and posterior element injuries with rotation) describe injury patterns resulting from axial torque. The latter are most often superimposed on either type A or type B lesions. Morphological criteria are predominantly used for further subdivision of the injuries. Severity progresses from type A through type C as well as within the types, groups, and further subdivisions. The 1445 cases were analyzed with regard to the level of the main injury, the frequency of types and groups, and the incidence of neurological deficit. Most injuries occurred around the thoracolumbar junction. The upper and lower end of the thoracolumbar spine and the T10 level were most infrequently injured. Type A fractures were found in 66.1%, type B in 14.5%, and type C in 19.4% of the cases. Stable type A1 fractures accounted for 34.7% of the total. Some injury patterns are typical for certain sections of the thoracolumbar spine and others for age groups. The neurological deficit, ranging from complete paraplegia to a single root lesion, was evaluated in 1212 cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Humans↗

PubMed1994

Quantitative MR imaging of lumbar intervertebral disc and vertebral bodies: methodology, reproducibility, and preliminary results.

Since relaxation times are influenced by the hydration of the tissue and the chemical environment of the water molecules, T1 and T2 measurements (quantitative MRI) could be used as an indicator for the water content and the biochemical composition of lumbar intervertebral discs. The discriminating power of quantitative MRI for tissue characterization in individuals (for clinical diagnosis) and in cohorts (e.g. for investigations on disc physiology or composition) relies on the reproducibility in relation to the expected tissue differences. We therefore investigated the reproducibility in vitro (lumbar spine phantom) and in vivo (10 volunteers). To estimate the differences between normal and pathologic tissues in vivo, 100 normal and 20 herniated intervertebral discs were examined by quantitative MRI in a first application of our method. The relaxation times were calculated from a set of 20 images obtained with five single-slice/multi-echo sequences at different TR values on a commercial whole-body system (1.5 T). We have found a satisfactory reproducibility in vitro (T1: 1.9%; T2: 6.2%), while the reproducibility was less satisfactory in vivo (T1: 16.4%; T2: 13.4%). Calculated from theses values, differences in relaxation times of various tissues must exceed 486 ms for T1 and 24 ms for T2 (tolerance limits) to allow discrimination with a 95% confidence in individuals. We observed statistically significant (p = 0.001) mean differences between normal (n = 100) and herniated (n = 20) intervertebral discs (delta T1: 196 ms; delta T2: 15 ms). Although statistical significant in cohorts, a discrimination of normal and herniated intervertebral discs is limited by quantitative MRI in individuals, since the differences are smaller than the tolerance limits necessary for a reliable clinical diagnosis. However, our results indicate that variations in the disc hydration and/or composition can be noninvasively detected by quantitative MRI in studies of cohorts with sufficient accuracy.

Adolescent↗

PubMed1994

Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast.

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.

Amino Acid Sequence↗

PubMed1994

[Single segment internal fixator device in treatment of thoracolumbar vertebral fractures].

Treatment of vertebral fractures by the fixed-angle system of the internal fixator allows fusion over a short area and is the form of treatment now in general use. A bisegmental construction is usual, which means that reduction and relief of the injured vertebral body is achieved indirectly by means of two intact vertebrae immediately adjacent on either side. As a result, both segments stiffen up or are permanently damaged in their function. We report on the possibility of single-segment fixation. The principle is the fusion of the injured segment itself only, i.e., the vertebra actually injured and the vertebra adjacent to the injured segment. Unnecessary fixation of a second healthy segment can thus be avoided. The characteristics of the fracture type in which single-segment stabilization is possible are explained. In a small study of the technique, we used the new AO classification of vertebral fractures of the thoracolumbar spine. The operation technique differs in some details from that applied with a multisegmental internal fixator. For example, the pedicle screws occasionally need to be inserted extremely close to the end-plates if the remaining part of the vertebral body has been destroyed and therefore cannot provide stability. Contraindications are broken pedicles and complete burst fractures of the body. With due consideration for these limitations fractures in all three main groups of the AO classification can, in principle, be stabilized in a single segment. In the years 1988-1990, 14 patients were operated on in this way (12 acute injuries, 2 secondary operations).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1993

Treatment of severe spondylolisthesis by reduction and pedicular fixation. A 4-6-year follow-up study.

Ten consecutive patients with severe spondylolisthesis were treated with reduction and pedicular fixation (four Internal Fixator instrumentations, six Cotrel-Dubousset instrumentations). There were six Grade III spondylolisthesis and four spondyloptoses at the L5/S1 level. All patients had complete clinical and radiographic evaluation with an average follow-up of 56 months (range, 43-75 months). The percentage of slippage averaged 78.5% preoperatively and 39.6% postoperatively. The slip angle averaged 43 degrees preoperatively and 17 degrees postoperatively. Four patients with spondyloptosis were treated with combined posterolateral and interbody fusion and had solid fusion without loss of reduction. Five of six patients in whom reduction and stabilization was performed by a single posterolateral fusion demonstrated loss of reduction, nonunion, and implant failure. Four of these patients were reoperated. Ultimately all patients had resolution of pain, solid fusion, and no further slip progression. Reduction, pedicular fixation, and combined posterolateral and interbody fusion is a technically demanding procedure, which should be reserved for selected patients. Pedicular fixation systems may only allow permanent reduction and stabilization of high-grade spondylolisthesis in conjunction with a combined interbody and posterolateral fusion.

Adolescent↗

PubMed1993

Anterior transpedicular fixation of the lower thoracic and lumbar spine. Experimental verification using a new direction finder.

Currently, no anterior spinal implant provides a strong bone-screw interface because of the cancellous characteristics of the vertebral body. A more secure anchorage could be obtained by anterior transpedicular screw fixation. Four hundred transpedicular screws located between T7 and L5 were placed using the newly developed direction finder. Measurements were obtained directly from radiographs of the cadaveric specimens. In 10 cases (2.5%), the screws crossed the medial pedicle border, but never by more than 1.4 mm. A lateral protrusion was noted in another 41 screws (10%), with no protrusion greater than 2.2 mm. Encroachments beyond the superior or inferior border were not observed. The mean angle of the screws at each level measured between 7 and 19 in the transverse plane and between 2 and 4.5 in the sagittal plane. This technique should be reserved for vertebrae without significant arthritic changes. The rare screw with minimal infraction through the medial or lateral pedicle wall should not cause any vascular or neural compromise. The anterior transpedicular screw technique appeared relatively safe (88%) and encouraged the development of the new plate system for anterior spinal stabilization.

Adult↗

PubMed1993

Nuclear PRP20 protein is required for mRNA export.

The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus.

Alleles↗

PubMed1993

Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.

Asparagine-linked N-glycosylation is an essential protein modification occurring in all eukaryotic cells. The central step is the co-translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn-X-Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum. This reaction is catalyzed by the enzyme N-oligosaccharyl transferase. In yeast, Wbp1p is an essential component of this enzyme. Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation. Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability. Similar to Wbp1p, depletion of Swp1p results in reduced N-oligosaccharyl transferase activity in vivo and in vitro. Wbp1p and Swp1p can be chemically cross-linked, suggesting that both proteins are essential constituents of the N-oligosaccharyl transferase complex.

Amino Acid Sequence↗

PubMed1993

Surgical treatment of tumors of the cervical spine and first two thoracic vertebrae.

From 1985 through 1990, 19 patients with tumorous conditions of the cervical spine and the first two thoracic vertebrae were treated with anterior, posterior, or combined anterior/posterior surgical techniques. Breast metastases were by far the most common condition (42%). Patients usually experienced severe pain, which resisted conservative treatment, sometimes associated with radiculopathies (42%) or neurological deficits (31%). To date, the treatment of spinal tumors is only palliative, and surgery must be considered for cases with unremitting neck pain, major vertebral destruction with loss, or impending loss of cervical spine stability and neurological deficits due to local tumor compression. Contrary to the commonly used posterior wiring stabilizations, we preferred stabilization techniques more closed to those used in traumatology. Our findings suggest anterior surgery alone with vertebrectomy and stabilization with plate and bone cement for tumors involving only one vertebra and localized between C3 and T1. Posterior approach and stabilization is advocated for atlantoaxial lesions. A combined anterior and posterior technique should be reserved for extended tumoral conditions where an anterior fixation does not offer enough stability or where more radical surgery is required. In the present series, immediate good spinal stabilization and neck pain relief was obtained in every case, allowing early mobilization. Improvement of the neurologic deficit was noted in 65% of our patients.

Adult↗

PubMed1993

Quantitative MR imaging of lumbar intervertebral disks and vertebral bodies: influence of diurnal water content variations.

The influence of diurnal water content variation on T1 and T2 and relative proton density (Nr) in intervertebral disks and vertebral bodies was studied in 10 healthy adult volunteers. T1, T2, and Nr were calculated from a set of 20 images obtained at 1.5 T with five single-section, multiecho sequences at different repetition times. Volunteers were randomized into study (one measurement in the morning and one in the evening) and control (two consecutive measurements in the morning) groups. In the study group, evening mean T1 and Nr in intervertebral disks and in vertebral bodies were significantly (P < .001) less than measured in the morning. Mean T2 in vertebral bodies significantly (P < .05) increased between morning and evening measurements. Diurnal variations were significantly less pronounced in degenerative than in normal intervertebral disks. No significant changes occurred in the control group. Relaxation time and Nr measurements allow sufficiently accurate estimation of diurnal water content variation in cohorts of vertebral bodies and intervertebral disks.

Adolescent↗

PubMed1993

[Follow-up and prognosis of neurologic sequelae of pelvic ring fractures with involvement of the sacrum and/or the iliosacral joint].

The extent of neurological lesions following an injury of the pelvic ring is often not initially recognized, as interest is then focused on the treatment of the pelvic ring fracture. Once the fracture has healed, the patient suffers from the sequelae of the neurological injury. Our series of 323 pelvic ring injuries includes 161 sacral fractures and 12 complete disruptions of the sacroiliac joint. Twenty-three patients sustained an injury of the lumbosacral plexus, and 20 patients were examined retrospectively. The different parts of the lumbosacral plexus showed variable recovery potential. An important or complete recovery was noted in 8 of 9 patients suffering from a motor deficit of the lumbar plexus, the obturator nerve, the superior gluteal nerve or the inferior gluteal nerve. Four out of 8 patients with a motor deficit of the sacral plexus had an important or complete improvement. In contrast to these results was the poor recovery of lesions of the lumbosacral trunk. Eight out of 11 patients showed no or only minor recovery, although the pelvic ring was stabilized by operative means in 9 patients. In 2 patients the lumbosacral trunk was directly decompressed by a dorsal approach. In both cases the recovery was complete. In 6 patients the sphincter function was damaged. Recovery was dependent on the localization of the sacral fracture. If the fracture traversed the sacral canal, no neurological improvement was noted.

Adult↗