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M Eckhardt

Publications and source records attributed to M Eckhardt.

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

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↗
PubMed1997

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1995

[Effect of calcium dobesilate on progression of diabetic retinopathy].

BACKGROUND: The increase of platelet aggregation, plasma and whole blood viscosity and the decrease of red cell deformability in patients with diabetes mellitus may be causative in the development of retinopathy. A therapy influencing these factors maybe prevents retinopathy or slows progression. MATERIAL AND METHODS: In a clinical and prospective study over 2 years 74 patients aged 20 to 69 years with a mild background retinopathy were randomly assigned to take oral 3 x 500 mg calcium dobesilate (Doxium) or no therapy. Both groups did neither differ in demographic nor in laboratory data. In fluorescein angiography following characteristics of retinopathy were graded: microaneurysms, size and outline of foveal avascular zone, capillary loss and severity of dye leakage. Additionally in 15 patients contrast sensitivity, visual fields and oscillatory potentials were investigated. RESULTS: The results of the fluorescein angiographical characteristics revealed no statistically significant difference between both groups. In the oscillatory potentials a significant shortening of latency could be found in both groups, but there was no difference between the groups. CONCLUSION: A 2-years application of calcium dobesilate has no statistically significant influence on the progression of diabetic retinopathy.

Adult↗
PubMed1995

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE.

Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.

Amino Acid Sequence↗
PubMed1994

Characterization of tumor-associated neural cell adhesion molecule in human serum.

In human serum, at least two molecular species of the neural cell adhesion molecule (NCAM) with molecular weights of 110,000-130,000 and 150,000-180,000, respectively, can be identified by Western blotting. Both are characterized by the absence of epitopes for monoclonal antibodies KD11 and MG5, which specifically recognize intracellular domains of the human NCAM transmembrane isoforms, NCAM-140 and NCAM-180. In contrast to the M(r) 110,000-130,000 molecule also detectable in serum samples from healthy blood donors, the M(r) 150,000-180,000 molecule appears to be tumor associated. The only difference between these two species is shown to be the presence of long chains of alpha-(2,8)-linked N-acetylneuraminic acids, which are characteristic for the so-called embryonic NCAM form. After treatment with endoneuraminidase N, the M(r) 150,000-180,000 molecule can no longer be discriminated from the M(r) 110,000-130,000 molecule in Western blotting as well as gel and anion exchange chromatography experiments. The experimental data clearly show that only the embryonic NCAM molecule carrying the poly-alpha-(2,8)-linked N-acetylneuraminic acid moiety can be regarded as a specific serum marker for small cell lung cancer.

Blotting, Western↗
PubMed1994

Hot spots of antigenicity in the neural cell adhesion molecule NCAM.

Monoclonal antibodies (MAbs) ranked together as small-cell-lung-cancer (SCLC) Cluster I MAbs are directed against the neural cell adhesion molecule NCAM (CD 56) and have been shown to be useful reagents in SCLC diagnosis and therapy. We analyzed the epitopes recognized by 5 SCLC cluster-I MAbs (123C3, 123A8, ERIC-I, MB2, and Leu 19) and a closely related anti CD 56 MAb (T199). Our results show that within the NCAM molecule Ig-like domain 3 and the segment of about 200 amino acids comprised by exons 11-13 are immunodominant regions. The MAbs investigated in this study can be combined into 2 groups. Group 1 consists of MAbs MB2, Leu 19 and T199, which are directed against epitopes located in the 3rd Ig-like domain. These MAbs recognize closely related but distinctive conformational epitopes. MAbs ERIC-1, 123C3 and 123A8 form Group 2 and are directed against a membrane-proximal region of the NCAM molecule. The data presented suggest that the 3 Group-2 MAbs bind to closely related or identical epitopes.

Adult↗
PubMed1994

[Heparin-induced extracorporeal LDL precipitation (H.E.L.P.). A new therapeutic possibility in vascular occlusion of the retina--initial results].

Heparin-induced extracorporeal LDL precipitation (HELP) eliminates selectively fibrinogen, LDL cholesterol, cholesterol, triglycerides and LP(a) from the blood plasma using extracorporeal circulation. The reduction of fibrinogen and LDL about 50% after only one procedure immediately improves the hemorrheological situation, which has so far not been achieved by any hemorrheologically active substance. Five patients (1 central retinal vein occlusion, 3 branch vein occlusions, 1 central arterial occlusion) ranging from 59-76 years underwent HELP treatment six times over a 5-week period. After the first HELP procedure the fibrinogen was lowered from 278 +/- 57.8 to 157 +/- 22.2 mg/dl and LDL from 162.2 +/- 58 to 82.4 +/- 32.5 mg/dl. Plasma viscosity decreased from 1.3 +/- 0.07 to 1.1 +/- 0.05 mPa/s. At the end of the treatment an increase in visual acuity of 3 or more lines and an improvement in the visual field was seen in all patients. Measurements of the arteriovenous passage time with laser scan video fluorescein angiography before and at the end of the therapy showed a significant decrease from 3.5 +/- 1.59 to 2.88 +/- 1.54 s (P < 0.05) without influencing the arm-retina time. This first clinical and hemorrheological data on the HELP procedure in retinal vessel occlusion demonstrate a promising regimen in the treatment of acute retinal microcirculatory diseases.

Aged↗
PubMed1991

New, long-acting, potent bradykinin antagonists.

1. Three new bradykinin (BK) antagonists, D-Arg0-Hyp3-Thi5-D-Tic7-Oic8-BK (compound I), D-Arg0-Hyp3-D-Tic7-Oic8-BK (compound II), and Arg(Tos)1-Hyp3-Thi5-D-Tic7-Oic8-BK (compound III), were tested against the effects of BK in 9 bioassay preparations including visceral smooth muscles, vasoconstriction, plasma protein extravasation, release of prostaglandin E2, bronchoconstriction, and stimulation of afferent C-fibre nociceptors. In some of these tests the effects of the new compounds were compared with those of the antagonist D-Arg0-Hyp2-Thi5,8-D-Phe7-BK (compound IV), described by Stewart & Vavrek (1987). 2. For all bioassays the general rank order of potency of the compounds was found to be I greater than II greater than III much greater than IV. The new antagonists were long-acting; in some bioassays their effects outlasted the duration of the experiment. 3. The inhibitory effects of the new BK antagonists were specific for BK; actions of noradrenaline, angiotensin II, acetylcholine or histamine were unaffected by the antagonists. They did not stimulate the release of histamine or prostaglandins. An agonistic effect was observed only with very high concentrations of compounds I and II in the plasma protein extravasation test. 4. The long duration of action of the new BK antagonists is probably due to a high and long-lasting affinity to the BK receptors. A high resistance of the antagonists to enzymatic destruction may be another reason. 5. The new BK antagonists will be valuable tools for the investigation of the pathophysiological role of BK. In addition they may offer a potential for therapeutic applications.

Afferent Pathways↗
PubMed1990

Demonstration of extrapulmonary activity of angiotensin converting enzyme in intact tissue preparations.

1. The activity of angiotensin converting enzyme (ACE) has been studied on functional parameters of intact isolated preparations of extrapulmonary tissues. The conversion of angiotensin I (A I) to angiotensin II (A II) and the cleavage of bradykinin (BK) were used as indicators of ACE activity. Captopril was employed as a specific inhibitor of ACE. 2. Captopril augmented the BK-induced contractions of the rat isolated uterus, the BK- and substance P-induced contractions of the guinea-pig ileum, and the BK-induced venoconstriction in the isolated perfused ear of the rabbit. Degradation of BK by ACE was calculated to be 52% in the rat uterus and 75% in the rabbit perfused ear. 3. Captopril inhibited the A I-induced contractions of the rat isolated colon, the A I-induced vasoconstriction in the isolated perfused ear of the rabbit and the rise in blood pressure induced by i.a. injections of A I in pithed rats. Conversion of A I to A II was calculated to be 13% in the rat colon and 26% in the rabbit perfused ear. 4. From estimations of the A II activity (bioassay on the rat colon) in the effluent of the perfused ear of the rabbit after injections of A I into the arterial inflow cannula it was calculated that approximately one tenth of A I was converted to A II during a single passage through the ear (less than 15 s). 5. The present experiments suggest that the high activity of ACE in endothelium of blood vessels of extrapulmonary tissues may provide an additional (endothelium-dependent) local vasoconstrictor mechanism by the rapid formation of A II and inactivation of BK. The ACE activity in non-vascular smooth muscles, other than those of blood vessels, may also affect the physiological functions of these tissues.

Angiotensin II↗