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M Eckhardt

Publications and source records attributed to M Eckhardt.

At least 19 recordsLinked to original sources

Mice deficient in the polysialyltransferase ST8SiaIV/PST-1 allow discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity.

Functional properties of the neural cell adhesion molecule (NCAM) are strongly influenced by polysialylation. We used gene-targeting to generate mice lacking ST8SiaIV/PST-1, one of the polysialyltransferases responsible for addition of polysialic acid (PSA) to NCAM. Mice homozygous for the null mutation reveal normal development of gross anatomical features. In contrast to NCAM-deficient mice, olfactory precursor cells in the rostral migratory stream express PSA and follow their normal pathway. Furthermore, delamination of mossy fibers in the hippocampal CA3 region, as found in NCAM-deficient mice, does not occur in ST8SiaIV mutants. However, during postnatal development these animals show a decrease of PSA in most brain regions compared to wild-type animals. Loss of PSA in the presence of NCAM protein but in the absence of obvious histological changes allowed us to directly address the role of PSA in synaptic plasticity. Schaffer collateral-CA1 synapses, which express PSA in wild types, showed impaired long-term potentiation (LTP) and long-term depression (LTD) in adult mutants. This impairment was age-dependent, following the time course of developmental disappearance of PSA. Contrary to NCAM mutant mice, LTP in ST8SiaIV mutants was undisturbed at mossy fiber-CA3 synapses, which do not express PSA in wild-type mice. The results demonstrate an essential role for ST8SiaIV in synaptic plasticity in hippocampal CA1 synapses, whereas PSA produced by different polysialyltransferase or polysialyltransferases at early stages of differentiation regulates migration of neural precursor cells and correct lamination of mossy fibers. We suggest that NCAM but not PSA is likely to be important for LTP in the hippocampal CA3 region.

Aging↗

Binding of Clostridium botulinum C2 toxin to asparagine-linked complex and hybrid carbohydrates.

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic subunit (C2I) capable of ADP-ribosylating actin and a binding subunit (C2II) that is responsible for interaction with receptors on eukaryotic cells. Here we show that binding of C2 toxin depends on the presence of asparagine-linked carbohydrates. A recently identified Chinese hamster ovary cell mutant (Fritz, G., Schroeder, P., and Aktories, K. (1995) Infect. Immun. 63, 2334-2340) was found to be deficient in N-acetylglucosaminyltransferase I. C2 sensitivity of this mutant was restored by transfection of an N-acetylglucosaminyltransferase I cDNA. C2 toxin sensitivity was reduced after inhibition of alpha-mannosidase II. In contrast, Chinese hamster ovary cell mutants deficient in sialylated (Lec2) or galactosylated (Lec8) glycoconjugates showed an increase in toxin sensitivity compared with wild-type cells. Our results show that the GlcNAc residue linked beta-1,2 to the alpha-1,3-mannose of the asparagine-linked core structure is essential for C2II binding to Chinese hamster ovary cells.

Animals↗

Membrane topology of the mammalian CMP-sialic acid transporter.

Nucleotide sugar transporters form a family of distantly related membrane proteins of the Golgi apparatus and the endoplasmic reticulum. The first transporter sequences have been identified within the last 2 years. However, information about the secondary and tertiary structure for these molecules has been limited to theoretical considerations. In the present study, an epitope-insertion approach was used to investigate the membrane topology of the CMP-sialic acid transporter. Immunofluorescence studies were carried out to analyze the orientation of the introduced epitopes in semipermeabilized cells. Both an amino-terminally introduced FLAG sequence and a carboxyl-terminal hemagglutinin tag were found to be oriented toward the cytosol. Results obtained with CMP-sialic acid transporter variants that contained the hemagglutinin epitope in potential intermembrane loop structures were in good correlation with the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to membrane-spanning regions inactivate the transport process, whereas those in hydrophilic loop structures have no detectable effect on the activity. This study points the way toward understanding structure-function relationships of nucleotide sugar transporters.

Amino Acid Sequence↗

Complete capsulorhexis opening occlusion despite capsular tension ring implantation.

An 89-year-old woman and an 86-year-old woman had continuous curvilinear capsulorhexis, phacoemulsification, and implantation of a silicone plate-haptic intraocular lens. Because of presumed weak zonules (high age, pseudoexfoliation), a poly(methyl methacrylate) capsular tension ring was also implanted. Despite this, both patients reported deterioration in visual acuity that was the result of complete occlusion of the anterior capsule opening by fibrotic tissue 4 and 3 months postoperatively, respectively.

Aged↗

Mutants of the CMP-sialic acid transporter causing the Lec2 phenotype.

Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker alpha-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter.

Animals↗

Mammalian cytidine 5'-monophosphate N-acetylneuraminic acid synthetase: a nuclear protein with evolutionarily conserved structural motifs.

Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is species- and tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMP-Neu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the lec32 mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.

Amino Acid Sequence↗

Intraocular pressure difference in Goldmann applanation tonometry versus Perkins hand-held applanation tonometry in overweight patients.

OBJECTIVE: To analyze the increase in intraocular pressure (IOP) caused by anatomic and physiologic factors in overweight patients when using Goldmann applanation tonometry. DESIGN: A prospective cohort study. PARTICIPANTS: Seventy average-weight individuals who had no difficulties with IOP measurements at the slit lamp and 12 obese patients with suspected glaucoma who could position the head at the slit lamp only with great effort participated. INTERVENTION: The authors compared IOP values between slit-lamp-mounted Goldmann applanation tonometry and Perkins hand-held tonometry. MAIN OUTCOME MEASURE: The difference in Goldmann and Perkins IOP measurements was examined. RESULTS: In the group of obese patients, the mean IOP was 20.9+/-2.28 mmHg (mean +/- standard deviation; range, 18-26 mmHg) for the right eye and 21.4+/-3.16 mmHg (range, 16-28 mmHg) for the left eye when determined by Goldmann tonometry and 16.3+/-2.39 mmHg (range, 13-20 mmHg) for the right eye and 16.3+/-2.42 (range, 11-19 mmHg) for the left eye when determined by Perkins tonometry. The mean decrease was 4.5+/-1.3 mmHg (range, 3-7 mmHg) for the right eye and 4.9+/-1.9 mmHg (range, 2-9 mmHg) for the left eye. In the control group, the mean difference between the two types of tonometers for the right eye was 0.34+/-0.69 mmHg and for the left eye was 0.33+/-0.82 mmHg. Patients who had a falsely elevated IOP on Goldmann tonometry had an average body mass index of 34+/-3.82 (range, 28.5-41.9); most were female (5:1 ratio). CONCLUSION: The authors believe simultaneous breath-holding and thorax compression, with subsequent increase in venous pressure, may be a causative factor for transitory elevations of IOP. Perkins tonometry in obese patients may help avoid a false diagnosis of glaucoma caused by transitory elevations in IOP.

Adult↗

Polysialic acid: three-dimensional structure, biosynthesis and function.

Polysialic acid is a unique cell surface polysaccharide found in the capsule of neuroinvasive bacteria and as a highly regulated post-translational modification of the neural cell adhesion molecule. Recent progress has been achieved in research on both the physicochemical properties of polysialic acid and the biosynthetic pathways leading to polysialic acid expression in bacteria and mammals.

Animals↗

Serum polysialylated neural cell adhesion molecule in childhood neuroblastoma.

Neuroblastoma cells express the polysialylated form of the neural cell adhesion molecule (NCAM), which normally becomes restricted to a few neural tissues after embryogenesis. In this study, we investigated serum levels of polysialylated NCAM in 14 children with different grades and stages of neuroblastoma using an immunoluminescence assay, and compared the results to 269 healthy control subjects. Simultaneously, the polysialylated NCAM content of the tumours was determined by immunohistochemistry. Serum levels were dramatically elevated (more than sixfold) in children with advanced stages and fatal courses of disease, whereas children with differentiated tumour types and limited disease had low or normal levels. Serum concentrations correlated with the polysialylated NCAM content of the tumours, and they decreased during successful therapy. We therefore suggest polysialylated NCAM to be a useful marker monitoring childhood neuroblastoma.

Biomarkers, Tumor↗

Genomic organization of the murine polysialyltransferase gene ST8SiaIV (PST-1).

Polysialic acid (PSA) is an important regulator of cellular interactions. Two enzymes (ST8SiaII and ST8SiaIV) are capable of synthesizing PSA. In the present study, the gene encoding the murine ST8SiaIV (PST-1) has been isolated and characterized. In contrast to the ST8SiaII (STX) gene which contains six exons and spans about 80 kb, the ST8SiaIV gene comprises only five exons spanning over at least 55 kb. However, alignment of the two genes revealed that exon-intron boundaries of exons 2-5 of ST8SiaIV and exons 3-6 of ST8SiaII are located at identical sites. Differences are restricted to the 5'-region encoded by one exon in the case of ST8SiaIV, whereas the corresponding region of ST8SiaII is interrupted by a very long intron. 5'-RACE analysis of the ST8SiaIV transcript using mRNA from AtT20 cells identified two transcription start sites at positions -324 and -204 relative to the translation start codon. The promoter region of ST8SiaIV lacks TATA- and CAAT-like sequences and is enriched in G+C (60%). The promoter contains putative Sp1, AP-1, AP-2, and PEA3 binding sites, as well as a purine- and a pyrimidine-rich region. Luciferase reporter gene assays demonstrated that the region between nucleotides -443 and -162 is sufficient to direct gene expression. The induction of luciferase activity was 30- and 10-fold in the PSA-positive AtT20 and CHO cells, but only 5- and 7-fold in the PSA-negative NIH-3T3 cells and in a PSA-negative subline of AtT20. Thus, although decreased in activity in PSA-negative cell lines, the basal promoter is not sufficient for the strong cell-type and tissue specific regulation of the ST8SiaIV gene, suggesting regulatory elements in the more upstream 5'-region.

Amino Acid Sequence↗

Pathogenesis of acute radiation effects in the urinary bladder. Experimental results.

PURPOSE: The present review summarizes experimental studies of the pathogenesis of acute radiation-induced changes in urinary bladder function. MATERIAL AND METHODS: Transurethral cystometry was used for longitudinal assessment of bladder function in mice. With this technique, radiation-induced changes in storage capacity can be quantified. In histological studies, changes in urothelial cell density and in urothelial protein expression during the acute radiation response were determined. Acetylsalicylic acid (ASA) was used for the treatment of acute functional changes. RESULTS: The histological studies did not reveal any systematic fluctuations in urothelial cell density during the time of the acute radiation response. However, characteristic changes in the expression of proteins associated with urothelial cell function, differentiation and cell contact were observed, which correlated with the functional impairment. By local or systematical application of ASA, a significant restoration of bladder function compared to placebo treatment could be achieved. CONCLUSION: Acute functional radiation effects in the urinary bladder are not based on urothelial denudation. However, changes in protein expression indicate an impairment of the urothelial barrier function. The results of ASA treatment demonstrate that prostaglandins are involved in the response. Alterations in urothelial or endothelial prostaglandin metabolism may be primarily radiation-induced or secondary because of the impaired urothelial barrier.

Animals↗

Molecular cloning of the hamster CMP-sialic acid transporter.

Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP-sialic acid transporter (CMP-Sia-Tr). Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMP-Sia-Tr which complements the Lec2 phenotype. The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP-Sia-Tr. The expression of a hamster CMP-Sia-Tr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter. Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3% similarity) of CMP-Sia-Tr to the recently cloned human UDP-galactose transporter (UDP-Gal-Tr). In contrast, sequence similarities to the yeast UDP-N-acetylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-mannose transporter (GDP-Man-Tr) of Leishmania donovani are restricted to a region encoding the two most C-terminally located transmembrane helices. A computer-based structural analysis of CMP-Sia-Tr proposes an eight transmembrane helix model with the N- and C-termini located on the cytosolic side of the Golgi membrane.

Amino Acid Sequence↗

Functional expression of the murine Golgi CMP-sialic acid transporter in saccharomyces cerevisiae.

We have functionally expressed the murine Golgi putative CMP-sialic acid transporter in Saccharomyces cerevisiae. Using a galactose-inducible expression system, S. cerevisiae vesicles were able to transport CMP-sialic acid. Transport was dependent on galactose induction and was temperature-dependent and saturable with an apparent Km of 2.9 microM. Transport was inhibited by CMP, and upon vesicle disruption with Triton X-100 parameters were very similar to the previously described CMP-sialic acid transport characteristics observed with mammalian Golgi vesicles. CMP-sialic acid transport induction was specific as no transport of UDP-galactose was observed even though the latter putative transporter has a high degree of amino acid sequence identity with the CMP-sialic acid transporter. Together, the above results demonstrate that the previously described cDNA encoding the putative CMP-sialic acid transporter encodes the transporter protein per se and suggests that this heterologous expression system may be used for further structural and functional studies of other Golgi membrane transporter proteins.

Animals↗

Autocatalytic polysialylation of polysialyltransferase-1.

Polysialic acid (PSA) is a specific and highly regulated post-translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase-1 (PST-1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST-1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST-1. The synthesis of PSA chains, which involved N-glycosylation sites, occurred immediately after contact with the activated sugar donor CMP-Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo-PST-1 isolated from CHO cells of the Lec2 complementation group. Pre-formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto-PST-1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST-1.

Animals↗

Polysialylation of NCAM by a single enzyme.

The addition of poly-alpha2,8-N-acetylneuraminic acid (polysialic acid; PSA) to the neural cell adhesion molecule NCAM plays a crucial role in neural development [1-3], neural regeneration [4], and plastic processes in the vertebrate brain associated with neurite outgrowth [5], axonal pathfinding [6], and learning and memory [7,-9]. PSA levels are decreased in people affected by schizophrenia [10], and PSA has been identified as a specific marker for some neuroendocrine and lymphoblastoid tumours [11-13]; expression of PSA on the surface of these tumour cells modulates their metastatic potential [11-13]. Studies aimed at understanding PSA biosynthesis and the dynamics of its production have largely been promoted by the cloning of polysialyltransferases (PST-1 in hamster; PST in human and mouse) [14-16]. However, the number of enzymes involved in the biosynthesis of PSA has not been identified. Using incompletely glycosylated NCAM variants and soluble recombinant glycosyltransferases, we reconstituted the site at which PST-1 acts to polysialylate NCAM in vitro. The data presented here clearly demonstrate that polysialylation of NCAM is catalyzed by a single enzyme, PST-1, and that terminal sialylation of the N-glycan core is sufficient to generate the PSA acceptor site. Our results also show that PST-1 can act on core structures with the terminal sialic acid connected to galactose via an alpha2,3 or alpha2,6 linkage.

Animals↗

Expression cloning of the Golgi CMP-sialic acid transporter.

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.

Amino Acid Sequence↗

Influence of a single HELP application on hemorheology and retinal hemodynamics.

The heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) eliminates selectively fibrinogen, total cholesterol, low-density lipoproteins (LDL) and triglycerides and improves hemorheology. We investigated the hemodynamic changes in 10 patients without ocular diseases performing a video fluorescein angiography after a single HELP procedure measuring the arm-retina time (ART) and the arteriovenous passage time (AVP). Laboratory parameters such as fibrinogen, cholesterol, LDL, triglycerides, red cell transit time, plasma and whole-blood viscosity were determined and retinal circulation was measured immediately before and after the HELP application. All of the laboratory parameters were lowered significantly. The ART was unaffected by the HELP procedure; however, there was a significant AVP reduction from 2.41 +/- 0.49 to 1.97 +/- 0.47 (p < 0.005). These results support the hemorheological and hemodynamic efficiency of the HELP system in microcirculatory diseases.

Adult↗

Molecular characterization of eukaryotic polysialyltransferase-1.

Polysialic acid (PSA) is a dynamically regulated product of post-translational modification of the neural cell adhesion molecule, NCAM. Presence of the large anionic carbohydrate modulates NCAM binding properties and, by increasing the intercellular space, influences interactions between other cell surface molecules. PSA expression underlies cell type- and developmental-specific alterations and correlates with stages of cellular motility. In the adult, PSA becomes restricted to regions of permanent neural plasticity and regenerating neural and muscle tissues. Recent data implicate its important function in spatial learning and memory, and in tumour biology. Here we describe the molecular characterization of polysialyltransferase-1, the key enzyme of eukaryotic PSA synthesis. In reconstitution experiments, the newly cloned enzyme induces PSA synthesis in all NCAM-expressing cell lines. Our data therefore represent convincing evidence that the polycondensation of alpha-2,8-linked sialic acids in mammals is the result of a single enzymatic activity and provide a new basis for studying the functional role of PSA in neuro- and tumour biology.

3T3 Cells↗