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Biomedical subjects

R Schauer

Publications and source records attributed to R Schauer.

At least 19 recordsLinked to original sources
PubMed2005

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca.

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6-0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac2, were detected in normal animals and made up 10-30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the alpha(2-6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.

Animals↗
PubMed2004

Effects of Pringle manoeuvre and ischaemic preconditioning on haemodynamic stability in patients undergoing elective hepatectomy: a randomized trial.

BACKGROUND: The Pringle manoeuvre and ischaemic preconditioning are applied to prevent blood loss and ischaemia-reperfusion injury, respectively, during liver surgery. In this prospective clinical trial we report on the intraoperative haemodynamic effects of the Pringle manoeuvre alone or in combination with ischaemic preconditioning. METHODS: Patients (n=68) were assigned randomly to three groups: (i) resection with the Pringle manoeuvre; (ii) with ischaemic preconditioning before the Pringle manoeuvre for resection; (iii) without pedicle clamping. RESULTS: Following the Pringle manoeuvre the mean arterial pressure increased transiently, but significantly decreased after unclamping as a result of peripheral vasodilation. Ischaemic preconditioning improved cardiovascular stability by lowering the need for catecholamines after liver reperfusion without affecting the blood sparing benefits of the Pringle manoeuvre. In addition, ischaemic preconditioning protected against reperfusion-induced tissue injury. CONCLUSIONS: Ischaemic preconditioning provides both better intraoperative haemodynamic stability and anti-ischaemic effects thereby allowing us to take full advantage of blood loss reduction by the Pringle manoeuvre.

Adult↗
PubMed2003

[52-year-old patient with subcutaneous space-occupying lesion in immunosuppression].

We report the case of a 52-years-old male patient, who was diagnosed with subcutaneous alveolar echinococcosis 6 months after liver transplantation for HCV-related cirrhosis. Nether the explanted nor the transplantated liver revealed an echinococcus focus. Therefore a rare primary extrahepatic manifestation was likely. Interestingly, the echinococcal larvae had developed protoscolices. The development of mature tapeworms in human is a rarity, which could be related to the immunosuppressive therapy after liver transplantation. The patient was curatively treated by surgical removal of the subcutaneous tumor and a postoperative therapy with albendazole. Furthermore, HCV reinfection (genotype 2b) was successfully treated with interferone alpha 2b and ribavirine for 6 months.

Biopsy↗
PubMed2003

Sialic acids are absent from the dermatophytes Trichophyton mentagrophytes and Trichophyton rubrum.

Experiments were performed to determine whether sialic acids are expressed in two dermatophytes: Trichophyton rubrum and T. mentagrophytes, similarly to other fungal pathogens. Chemical extraction of mycelia and microconidia followed by high-performance thin-layer chromatography and colorimetric assays were all negative for sialic acid. Incubation of dermatophytes in the presence of Limax flavus agglutinin, specific for sialic acid, was negative in a fluorescence staining test. We have also used other lectins that bind to sialic acid and/or other sugar residues, and these ligands coupled to fluorescein strongly stained these fungi. Such fluorescence staining was not abolished or reduced when fungi were pretreated with sialidase. Different strains of influenza virus which recognize sialic acid residues were also used, but no agglutination of the dermatophytes was observed. Based on these methods, which successfully revealed the presence of sialic acids in other fungal pathogens, we show that these monosaccharides do not occur in both dermatophyte species. Thus, sialic acids do not seem to play a role in the pathogenicity of these fungi.

Agglutination↗
PubMed2002

[Surgical therapy of advanced gallbladder carcinoma].

The use of surgery for the treatment of advanced gall bladder cancer is controversially discussed. This retrospective study included 204 patients who were subjected to surgery due to advanced gall bladder cancer at the Klinikum Grosshadern. Mean survival time of all patients was 4.5 months. Advancement of the tumor stage resulted in a decreased percentage of possible R0 resections (T3 n = 48, R0 31%, T4 n = 87, R0 13%). Nonetheless, R0 resections of T3 tumors significantly increased the survival rate compared to R1 and R2 resections (mean survival 20.2 vs. 4.5 months). R0 resections of T4 tumors also significantly attenuated the survival rate (18.1 vs. 2.4 months compared to R1 and R2 resections). Thus, diagnostic procedures have to focus on identifying patients with possible R0 resections and perform extensive resections on those patients.

Cholecystectomy↗
PubMed2001

Fibronectin is a binding partner for the myelin-associated glycoprotein (siglec-4a).

The myelin-associated glycoprotein (MAG) mediates cell-cell interactions between myelinating glial cells and neurons. Here we describe the extracellular matrix glycoprotein fibronectin as a binding partner of MAG. It has been identified by affinity precipitation with MAG-Fc from NG108-15 cells and by microsequencing of two peptides derived from a 210-kDa protein band. Western blot analysis showed that fibronectin is also present in MAG binding partners isolated from N(2)A (murine neuroblastoma) cells, rat brain and rat spinal cord. Different fibronectin isoforms have been isolated from brains of young and adult rats, indicating that the expression of MAG binding fibronectin changes during development.

Animals↗
PubMed2001

[Dissection techniques in liver surgery].

The first liver resection was performed in 1888. Since then a wide variety of dissection techniques have been introduced. The blunt dissection was replaced by novel methods, i.e. the CUSA technique and the Jet Cutter for major liver resections. These methods represent selective dissection techniques; whereas non-selective methods include the scalpel, scissors, linear stapling cutter, high-frequency coagulation, and the laser technique. The aim of this review article is the comparison of the different resection techniques in liver surgery, focussing on blood loss and resection time.

Blood Loss, Surgical↗
PubMed2001

[The efferent lacrimal ducts of the human. Morphological and biochemical studies].

PURPOSE: Infections occur frequently in the region of the efferent tear ducts. Exact knowledge of the anatomical structure and of cellular defense mechanisms is necessary to understand the pathological processes. This study analyzed the efferent tear ducts with regard to physiological function and possible defense mechanism against infections. MATERIALS AND METHODS: We used histological, immunohistochemical and electronmicroscopic techniques to investigate the lacrimal systems from 31 body donors aged 54-88 years. RESULTS: The efferent tear ducts are lined by a double-layered epithelium resting on a broad basement membrane. These cells contain many lipid droplets and secretory vacuoles in their apical part. Inside the epithelium cells are arranged partly in cell groups forming mucous glands, which morphologically resemble goblet cells of the tarsus palpebrae. The secretory products of these cells contain various carbohydrates including sialic acid. Lymphocytes and macrophages are found in the submucosa partly penetrating the epithelium. CONCLUSIONS: Lipids and mucins of epithelial and goblet cells form a specialized protective layer on the epithelium of the tear ducts which enables easy drainage of tear fluid into the inferior meatus of the nose. Together with immunocompetent cells the protective layer prevents invasion of pathogenetic agents.

Aged↗
PubMed2001

N-Glycolylneuraminic acid in human tumours.

N-Glycolylneuraminic acid (Neu5Gc) is an abundant sialic acid, occurring in the glycoconjugates of most deuterostome animals. Homo sapiens is a notable exception, since Neu5Gc is effectively absent from normal human tissues. This is due to a deletion in the human gene coding for CMP-Neu5Ac hydroxylase, the enzyme usually responsible for Neu5Gc biosynthesis. Despite this mutation, persistent reports in the literature suggest that Neu5Gc occurs in the glycoconjugates of many human tumours, where it might be responsible for the formation of so-called Hanganutziu-Deicher antibodies. However, the variety of systems studied and the various experimental approaches adopted have yielded a complex picture of Neu5Gc occurrence in human neoplasias. The aim of this paper is therefore to provide a critical review of the evidence for Neu5Gc in human tumours, paying particular attention to the analytical methods employed. The possible clinical applications of Neu5Gc-containing glycoconjugates and Hanganutziu-Deicher antibodies in the diagnosis and treatment of breast cancer and melanoma are also discussed. In view of the lack of CMP-Neu5Ac hydroxylase in human cells, alternative metabolic pathways for the biosynthesis of glycoconjugate-bound Neu5Gc are considered.

Antibodies, Heterophile↗
PubMed2001

Cloning and expression of a membrane-bound CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens.

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesized by the action of CMP-Neu5Ac hydroxylase. The enzyme from various mammals has been purified, characterized and sequenced by cDNA cloning. Although functional sequence motifs can be postulated from comparisons with several enzymes, no global homologies to any other proteins have been found. The unusual characteristics of this hydroxylase raise questions about its evolution. As echinoderms are phylogenetically the oldest organisms possessing Neu5Gc, they represent a starting point for investigations on the origin of this enzyme. Despite many similarities with its mammalian counterpart, CMP-Neu5Ac hydroxylase from the starfish A. rubens exhibits fundamental differences, most notably its association with a membrane and a requirement for high ionic strength. In order to shed light on the structural basis for these differences, the primary structure of CMP-Neu5Ac hydroxylase from A. rubens has been determined by PCR and cDNA-cloning techniques, using initial sequence information from the mouse enzyme. The complete assembled cDNA contained an ORF coding for a protein of 653 amino acids with a molecular mass of 75 kDa. The deduced amino-acid sequence exhibited a high degree of homology with the mammalian enzyme, although the C-terminus was some 60 residues longer. This extension consists of a terminal hydrophobic region, which may mediate membrane-binding, and a preceding hydrophilic sequence which probably serves as a hinge or linker. The identity of the ORF was confirmed by expression of active CMP-Neu5Ac hydroxylase in E. coli at low temperatures.

Amino Acid Sequence↗
PubMed2001

The sialate-pyruvate lyase from pig kidney. Elucidation of the primary structure and expression of recombinant enzyme activity.

The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments. The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template. The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme. The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes. The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group. In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%). Thirty-one amino acids were found to be absolutely conserved in all lyase sequences. Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction. After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences. After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.

Amino Acid Sequence↗
PubMed2001

Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens: insights into the reaction mechanism.

The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography. The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 degrees C. In kinetic studies the lyase exhibits a Km of 3.2 mM for Neu5Ac and a Vmax of 27.5 U x mg(-1). To clarify the functional role of some putative active site residues, site-directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate. Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding. In this respect the carboxy group of aspartate 187 seems to be particularly important. Based on the results of these studies, a model of the reaction mechanism is discussed.

Amino Acid Substitution↗
PubMed2001

Detection of sialic acids and gangliosides with special reference to 9-O-acetylated species in basaliomas and normal human skin.

Basal cell carcinomas and normal skin were examined in relation to the abnormal expression of gangliosides. The content of gangliosides with 9-O-acetylated sialic acids of 26 sample pairs was analyzed by a microtiter assay using influenza C virus as well as by fluorimetric high-performance liquid chromatography of the sialic acids released. The 9-O-acetylation levels were significantly (up to 56-fold) higher in basal cell carcinoma tissues than in the skin surrounding basal cell carcinomas. Slightly elevated amounts of O-acetylated gangliosides were also seen in the skin marginal to the basaliomas. The ganglioside composition of four sets of pooled samples of basal cell carcinoma and one pool of normal skin were studied by high-performance thin-layer chromatography and immune high-performance thin-layer chromatography using monoclonal antibodies against 9-O-acetyl GD3. The lipid-bound sialic acid content of normal skin was 0.029 microg dry weight, whereas in nodular basal cell carcinomas it was approximately twice as much. Several O-acetylated sialic acids were seen by high-performance liquid chromatography analysis, but N-acetyl-9-O-acetylneuraminic acid prevailed. Only in the tumor ganglioside fraction, a small amount of N-glycolylneuraminic acid was found. The 9-O-acetylated gangliosides with mainly 9-O-acetyl-GD3 can be considered as tumor-associated antigens or markers for basal cell carcinomas. This finding about tumor-associated carbohydrates may contribute to new strategies in current tumor diagnosis and therapy.

Acetylation↗
PubMed2001

[Surgical techniques in hepatic resections: Ultrasonic aspirator versus Jet-Cutter. A prospective randomized clinical trial].

AIM OF THE STUDY: For all resection-techniques of liver tissue intra- and post-operative blood-loss remains an important problem. Two novel resection-techniques the ultrasound-aspirator (CUSA) and the water-jet dissector (Jet-Cutter) appear to offer significant advantages regarding this problem. Aim of the present prospective clinical study was the comparison of these dissection techniques. MATERIAL AND METHODS: Prospective randomized study with the end points blood-loss, length of surgery, tissue trauma and long-term survival. FINDINGS: Significant differences between both procedures with Jet-Cutter (n = 31) versus ultrasonic surgical aspirator CUSA (n = 30) were observed regarding length of resection and complete liver ischemia time (Pringle-time). Here significant advantages of the jet-cutter-technique were observed with 28 +/- 11 minutes length of resection versus 46 +/- 19 minutes and 29 +/- 12 minutes Pringle-time versus 39 +/- 16 minutes. Furthermore, significant fewer blood transfusions were required following jet-cutter-resection with a mean of 1.5 blood units vs. 2.5 blood units using the CUSA. No differences were observed regarding postoperative long-term survival. CONCLUSIONS: The jet-cutter-technique is a fast and safe surgical procedure for liver resections and offers an attractive therapeutic alternative for various indications in liver surgery.

Blood Loss, Surgical↗
PubMed2001

Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes.

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.

Animals↗
PubMed2001

Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives.

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.

Acylation↗